Adsorption of pathogenic prion protein to quartz sand

Management responses to prion diseases of cattle, deer, and elk create a significant need for safe and effective disposal of infected carcasses and other materials. Furthermore, soil may contribute to the horizontal transmission of sheep scrapie and cervid chronic wasting disease by serving as an environmental reservoirforthe infectious agent. As an initial step toward understanding prion mobility in porous materials such as soil and landfilled waste, the influence of pH and ionic strength (l) on pathogenic prion protein (PrPsc) properties (viz. aggregation state and zeta-potential) and adsorption to quartz sand was investigated. The apparent average isoelectric point of PrPsc aggregates was 4.6. PrPsc aggregate size was largest between pH 4 and 6, and increased with increasing l at pH 7. Adsorption to quartz sand was maximal near the apparent isoelectric point of PrPsc aggregates and decreased as pH either declined or increased. PrPsc adsorption increased as suspension l increased, and reached an apparent plateau at l approximately 0.1 M. While trends with pH and l in PrPsc attachment to quartz surfaces were consistent with predictions based on Born-DLVO theory, non-DLVO forces appeared to contribute to adsorption at pH 7 and 9 (l = 10 mM). Our findings suggest that disposal strategies that elevate pH (e.g., burial in lime or fly ash), may increase PrPsc mobility. Similarly, PrPsc mobility may increase as a landfill ages, due to increases in pH and decreases in l of the leachate.     

Environmentally-relevant forms of the prion protein

Scrapie and chronic wasting disease (CWD) are prion diseases of particular environmental concern as they are horizontally transmissible and can remain infectious after years in the environment. Recent evidence suggests that the N-terminus of PrPSC, the infectious conformation of the prion protein, plays an important role in the mechanism of sorption to soil particles. We hypothesize that, in a prion-infected animal carcass, a portion of the N-terminus of PrPSc could be cleaved by proteinases in the brain at ordinary temperatures. Hamster (HY transmissible mink encephalopathy-infected), transgenic mice (CWD-infected), and elk (CWD-infected) brain homogenates were incubated at 22 and 37 degrees C for up to 1 month and then analyzed by Western blot using N-terminal and middle region monoclonal anti-PrP antibodies. For all three systems, there was a very faint or undetectable N-terminal PrP signal after 35 days at both temperatures, which indicates that full-length PrPSc might be rare in the brain matter of animal carcasses. Future studies on prion-soil interactions should therefore consider N-terminal-degraded PrPSc in addition to the full-length form. Both mouse and elk CWD PrPSc demonstrated greater resistance to degradation than HY TME PrPSc. This indicates that the transgenic mouse-CWD model is a good surrogate for natural CWD prions, but that other rodent prion models might not accurately represent CWD prion fate in the environment.     

Effects of Electrical Stimulation on the Functional Properties of Lamb Muscle

One side from each of eleven lamb carcasses was electrically stimulated (60 Herz alternating current at 240v followed by 420v) within 45 min postmortem. Three carcasses (stimulated and respective control sides) were rapidly chilled (1°C), three carcasses received a delayed chill (12°C until pH 6.0 was attained, then excision and 1°C), and three carcasses were slowly chilled (12°C). Measurements beginning at 44 hr postmortem showed that stimulation increased the sarcomere lengths but had few consistent or significant effects on water-holding capacity, protein solubility, emulsifying capacity, gel strength, cooking loss, or binding strength. The remaining two carcasses were rapidly chilled and their meat was manufactured into frankfurters. The emulsion stability was slightly improved by stimulation but smokehouse weight loss, Warner-Bratzler shear force, penetration force, cooking loss, and sensory characteristics were unaffected.     

Fate of prions in soil: adsorption and extraction by electroelution of recombinant ovine prion protein from montmorillonite and natural soils

Prions, the infectious agents thought to be responsible for transmissible spongiform encephalopathies, may contaminate soils and have been reported to persist there for years. We have studied the adsorption and desorption of a model recombinant prion protein on montmorillonite and natural soil samples in order to elucidate mechanisms of prion retention in soils. Clay minerals, such as montmorillonite, are known to be strong adsorbents for organic molecules, including proteins. Montmorillonite was found to have a large and selective adsorption capacity for both the normal and the aggregated prion protein. Adsorption occurred mainly via the N-terminal domain of the protein. Incubation with standard buffers and detergents did not desorb the full length protein from montmorillonite, emphasizing the largely irreversible trapping of prion protein by this soil constituent. An original electroelution method was developed to extract prion protein from both montmorillonite and natural soil samples, allowing quantification when coupled with rapid prion detection tests. This easy-to-perform method produced concentrated prion protein extracts and allowed detection of protein at levels as low as 0.2 ppb in natural soils.     

Physical and sensory attributes of hot-processed and conventionally chilled hams and bellies from electrically stimulated and non-stimulated pork carcasses

Forty-eight fresh hams and bellies were obtained from 24 market weight hogs (x = 94·5 kg) of which twelve were electrically stimulated (ES) by pulsing current immediately after exsanguination. The left side of each non-stimulated (NS) carcas was fabricated after conditioning for 3h post mortem at 17°C (NS hot-processed). The left sides of ES carcasses were fabricated 1 h pm. The right sides were fabricated following a 24 h cooler chill at 2°C (conventionally chilled: CP). Hams from ESCP carcasses had higher (P < 0·05) smokehouse yields than hams from NS carcasses. Hams that were hot-processed had higher smokehouse yields than the NSCP hams. Time of fabrication (1, 3 or 24h post mortem) did not affect smokehouse yields. Conventionally chilled bellies obtained from ES carcasses showed higher (P < 0·05) residual nitrite levels than those front electrically stimulated hot-processed (ESHP) carcasses. No differences were found for residual nitrite levels in the non-electrically stimulated sides. Panelists were unable to detect any sensory differences from the bacon strips. Sensory scores of ham slices were more juicy for non-stimulated hot-processed carcasses (NSHP) than those from ESHP carcasses. Panelists found the ham slices from NSCP carcasses to be more tender (P < 0·05) than those from electrically stimulated cold-processed (ESCP) carcasses. Results from this study clearly indicated that hot-processing of pork can provide hams and bellies that are acceptable for the production of cured hams and bacon of comparable quality and yield to those currently being produced under conventional processing methods.     

Inhibition of protease activity. Part 1. The effect on tenderness and indicators of proteolysis in ovine muscle

To examine the effect of particular enzyme groups on tenderness specific cysteine protease inhibitors were injected into muscle early post-mortem. The protease enzyme inhibitor E-64 was injected into the m. longissimus thoracis et lumborum (LTL) on the right side of 12 lamb carcasses within 15 min of death and in another 12 carcasses with the protease inhibitor Z-Phe-Ala-CHN(2). The left LTL (control) was injected with saline (0.25 M NaCl). To create variation in the rate of pH decline alternate carcasses were electrically stimulated (low voltage). The LTL was divided into cranial and caudal portions and aged for 1 or 2 days. Muscle samples at 1 day post-mortem were used for measurement of osmolality and sarcomere length (n=48), and others at 1 and 2 days post-mortem for shear force determination (n=96). The myofibrillar fragmentation index (MFI) was determined on samples taken at pH 6.2 and 1 and 2 days post-mortem (n=144). Other muscle samples were obtained at death, pH 6.2 and 6.0 and then at 1 and 2 days post-mortem (n=215). These samples were used for determination of protein solubility and the concentration of free amino acids. Stimulation caused a faster (P<0.05) decline in pH. There was no effect of stimulation (P>0.05) on shear force values, but injection of inhibitor and ageing both had effects (P<0.001). The inhibitor E-64 prevented any improvement in tenderness with ageing, whereas the inhibitor Z-Phe-Ala-CHN(2) and the control samples showed a similar ageing response. In the latter two treatments there was an average reduction of 1 kg in shear between 1 and 2 days post-mortem, whilst the inhibitor E-64 maintained shear force on average 2 kg higher than control samples. Injection and ageing had an effect on MFI (P<0.001) and there was an interaction (P<0.05) between stimulation and ageing for MFI, such that as stimulated muscle aged the rate of change of MFI was greater. There was an interaction between injection and ageing (P<0.05) for protein solubility such that samples treated with E-64 showed a minimal increase in protein solubility with ageing, whereas in samples treated with Z-Phe-Ala-CHN(2) and the control samples there was a significant increase. There was also an interaction between stimulation and ageing such that between sampling at pH 6.0 and 2 days post-mortem, stimulated muscle exhibited greater solubility (P<0.05). There were no effects (P>0.05) on the concentration of free amino acids. The evidence indicated that the cysteine proteases were responsible for post-mortem proteolysis and tenderisation, in particular the calpains, whereas the cathepsins (B and L) were unlikely to contribute to proteolysis and subsequent tenderisation in meat.     

The relationship between post-mortem calcium concentration or pH and indicators of proteolysis in ovine muscle

The relationship between post-mortem calcium concentration in muscle and indicators of proteolysis was examined and compared with pH as a predictor of these changes. Muscle samples (m. longissimus et thoracis lumborum; LTL) were obtained from the left side of 24 lamb carcasses at pH 6.2 and 6.0 and then at 1 and 2 days post-mortem (n=96). Alternate carcasses were electrically stimulated (low voltage) within 15 min of death. The myofibrillar fragmentation index (MFI) was determined on samples taken at pH 6.2 and 1 and 2 days post-mortem (n=72). Protein solubility, pH and free calcium concentration were measured on all samples (n=96). The post-mortem degradation of myofibrillar proteins was followed using SDS electrophoresis on all samples (n=96) from which the relative amounts of the 30 kDa fragment and a protein designated M1 were quantified. Transformation of data values for each variable (MFI, protein solubility, 30 kDa fragment and protein M1) improved the normality of the residuals and increased the variance explained by either calcium concentration or pH. pH was a better predictor of MFI and the 30-kDa fragment than calcium concentration and this was reversed when protein solubility and the protein designated M1 was predicted. Of the variables protein solubility could be predicted with the greatest accuracy using calcium concentration (R²=0.64; R.S.D.=0.81) or pH (R²=0.60; R.S.D.=0.85). However overall calcium concentration was not superior to pH as a predictor of the changes in the different indicators of proteolysis examined in this study.     

Effects of Certain Electrical Stimulation Parameters on Quality and Palatability of Beef

Grain-finished steers and heifers (n=390) were slaughtered in a commercial plant and assigned to one of five groups: (a) electrical stimulation (ES) immediately after exsanguination, (b) ES immediately before evisceration, (c) ES immediately after evisceration, (d) ES immediately after splitting, and (e) untreated (not ES). ES consisted of either 150 or 550 volts for either 1 or 2 mm. Electrically stimulated carcasses had more youthful, brighter colored lean and less “heat-ring” than untreated (not ES) carcasses. Steaks from electrically stimulated carcasses had (P<0.05) lower shear force values, were more tender, had less panel-detectable connective tissue and higher overall palatability ratings than did steaks from untreated (not ES) carcasses. Steaks from carcasses receiving 550-volt ES usually had more youthful, brighter colored lean and lower shear force values, were more tender, had less panel-detectable connective tissue and higher overall palatability ratings than steaks from carcasses that received 150-volt electrical stimulation. Quality and palatability traits of carcasses electrically stimulated for 1 mm were not different from those of carcasses receiving 2 mm of electrical stimulation. Electrical stimulation at different stages in the slaughter-dressing sequence did not evoke differential responses in quality or palatability factors.     

Early postmortem electrical stimulation simulates PSE pork development

Carcasses from 64 gilts were subjected to electrical stimulation (ES) at 3, 15, 25, 35, 45, and 55 min postmortem or were untreated (NS). Temperature and pH of longissimus muscles were recorded at 1, 7, 14, 20, 30, 40, 50, and 60 min, and 24 h postmortem. Muscle samples were collected at 1, 30 and 60 min, and 24 h for determining glycolytic metabolite concentrations. ES at 3, 15, and 25 min resulted in lower (P<0.05) muscle pH, but stimulation after 25 min had no effect on muscle pH. Likewise, ES prior to 25 min resulted in greater (P<0.05) muscle temperatures. Muscle lactate concentrations were greater (P<0.05) in carcasses stimulated before 45 min postmortem. Glucose 6-phosphate concentration decreased (P<0.05) during the first hr postmortem and increased (P<0.05) thereafter. ES of carcasses at 45 and 55 min resulted in higher (P<0.05) concentrations of muscle glucose 6-phosphate at 24 h compared with NS and early-stimulated carcasses. Muscle glycogen concentrations at 30 min in carcasses stimulated at 3, 15 and 25 min were lower (P<0.05) than NS carcasses. Carcasses stimulated at 3 and 15 min exhibited lower (P<0.05) concentrations of muscle glycogen at 60 min than NS carcasses. Carcasses stimulated at 3 and 15 min postmortem exhibited lower (P<0.05) color and firmness scores, while ES at 3 and 25 min postmortem resulted in lower (P<0.05) water holding capacity. ES had no significant effect on CIE L(?), a(?), b(?), or 24 h muscle pH. These data show that ES of pork carcasses during the first 25 min postmortem creates PSE-like quality characteristics and suggest that ES is a potential model for studying pork quality development.     

The electrical stimulation of beef carcasses

Beef carcasses were stimulated shortly after slaughter at varying voltage, pulse frequency and duration, with the aim of accelerating the fall of pH, destruction of ATP and onset of rigor in the muscles, so that the meat could be chilled rapidly after slaughter without danger of cold-shortening. Optimal effects were produced at 700 V, 25 Hz for 2 min, to give a total of 3000 pulses. In undressed carcasses stimulation induced a pH fall to 6.0 within 1 h of slaughter and to 5.7 within 2.5 h in the major muscles of the forelimb, back and thigh, representing a gain of more than 8 h over the time required in unstimulated carcasses hanging at 16°C. Similar results were obtained with dressed carcasses and sides, after allowing for the 50 min or so lost in dressing. The apparently high rate of pH fall from 6.3 to 5.7 after stimulation had ceased, compared with that over the same pH range in unstimulated carcasses, can be attributed mainly to the different muscle temperatures in the two cases (38 and 26°C respectively, in four major muscles). The muscles of stimulated carcasses showed no deleterious effects of stimulation. The observed drip loss from the hindlimb jointed 6 days after slaughter was not significantly greater than that from unstimulated carcasses.     

Electrical Stimulation of Mature Cow Carcasses and Its Effect on Tenderness, Myofibril Protein Degradation and Fragmentation

The effect of electrical stimulation was studied in the semi-membranosus, longissimus dorsi and triceps brachii of cow carcasses. Only one side of each carcass was stimulated (300V a.c., 120 sec). In each muscle, temperature, pH and myofibril fragmentation index (MF1) were measured and SDS-polyacrylamide gel electrophoresis performed at 0, 3, 6, 24, 72, and 168 hr post-stimulation while steaks for shear test were taken at 24, 72, and 168 hr. Electrical stimulation resulted in higher MF1, lower pH, higher temperature and lower shear force compared to the control but the time of appearance of the 30,000 dalton protein was not affected. Electrical stimulation improves tenderness by myofiber disrupture.     

Functionality of high and low voltage electrically stimulated beef chilled under moderate and rapid chilling regimes

Chuck muscles from 24 beef carcasses electrically stimulated (ES) with either high or low voltage, or a combination of both, and submitted to conventional or rapid chilling regimes were used in a model system study (pH, salt-solubloe protein extraction, emulsifying capacity) and in frank-further fabrication and analysis (yield, color, texture). Glycolytic rates measured in the loin had no effects on the functional parameters or on the quality of frankfurters. The ultimate pH values of unstimulated carcasses remained higher than in any ES carcasses, and led to higher protein extraction (P ≤ 0·05). However, ES had no further influence on the emulsifying capacity or on frankfurter yield and quality parameters independently of the voltage used (P > 0·05). Chilling regimes had no influence on the functional parameters of the model system but slightly influenced the yield and chewiness of the frankfurters (P ≤ 0·05). Therefore, the use of any type of ES in combination with either conventional (Canadian) or more rapid chilling is unlikely to have commercial significance on the yield or quality of frankfurters.     

Comparison of stunning methods in the dissemination of central nervous system tissue on the beef carcass surface

Beef carcasses were examined to explore the effects of stunning methods on central nervous system tissue (CNST) dissemination on the surface during the slaughtering process. The frequency of occurrence of CNST contamination on four defined parts, each on the interior and exterior surfaces of the split carcass, and their level of the contamination were measured by an ELISA test. The effect of hot carcass weight was also examined. The results showed that the frequency of contamination occurrence was not affected by the stunning method. However, the penetrating captive bolt stunning method resulted in a higher level of CNST contamination than non-penetrating sledge-hammer stunning (P < 0.001). The higher level of contamination occurred on the interior surface of carcasses along the vertebral area. Therefore, the dissemination of CNST on carcasses seemed to be affected by the stunning method, carcass splitting, and carcass washing. The carcass weight significantly affected the level of CNST contamination on the carcass (P < 0.01) but there was no interaction with the stunning method.     

The effect of ultra-rapid chilling and subsequent ageing on the calpain/calpastatin system and myofibrillar degradation in lamb M. longissimus thoracis et lumborum

The effect of ultra-rapid chilling (-20°C) on the proteolytic degradation of myofibrillar proteins during ageing, by the calpain/calpastatin system in lamb was investigated. Carcasses (36) were ultra-rapidly chilled (-20°C for 3.5 h followed by 20.5 h at +4°C) or chilling at +4°C for 24 h. After chilling all carcasses were stored at +4°C for a further 4 days. Samples of M. longissimus thoracis et lumborum were taken for Warner-Bratzler shear force measurement, sarcomere length, SDS-PAGE and enzyme activity analysis. Ultra-rapid chilling (-20°C) had no effect on the proteolytic degradation of myofibrillar proteins by the calpain/calpastatin system compared to conventional chilling (+4°C). However, no difference in tenderness was found between the two chilling regimes at 1 or 5 days postmortem so differences in myofibrillar degradation or enzyme activity would not be expected.     

Effect of electrical stimulation on protease activity and tenderness of M. longissimus from cattle with different proportions of Bos indicus content

The effect of electrical stimulation on protease activity (at approx. 3 h postmortem), sensory tenderness scores and shear force was determined on M. longissimus samples from three Bos indicus genotypes (0% Hereford, 50% Brahman×Hereford and 100% Brahman). The samples were divided and aged for 1 or 30 days. Electrical stimulation resulted in a general reduction in calpastatin activity suggesting that it accelerated proteolysis. Calpastatin activity increased commensurate with increasing Bos indicus content. Several significant interactions were shown, the most relevant of these was the interaction between Bos indicus content×electrical stimulation. In contrast to the other genotypes, calpain I and calpain II activities were shown to increase (significant for calpain II only) following stimulation in the purebred Brahmans (100%). There was a significant reduction in tenderness with increasing Bos indicus content. However, breed differences in shear force were reduced by electrical stimulation. The improvement in shear force following ageing was smaller for stimulated carcasses compared to the controls. This tends to reinforce the premise that electrical stimulation accelerates proteolysis. The results of this study show clear genotypic differences in proteolytic activity and tenderness. However, electrical stimulation can be employed to reduce breed differences in tenderness of the M. longissimus.     

The tenderising effect of electrical stimulation of beef carcasses

Electrical stimulation (ES) of beef carcasses soon after death has an accelerated tenderising effect on the musculature, under conditions of slow cooling (8 h at 16°C and then storage in still air at 1°C). The effect is large in the LD muscles, reducing the shear force on day 1 of storage from 11 to 6 kg; on day 14, the difference is still 3·3 kg. These differences would be detected by taste panels. The St muscles show a similar, but less pronounced, effect which would probably not be detected by taste panels. The accelerated tenderisation due to ES can be accounted for by the higher temperatures obtaining in stimulated muscles at the onset of rigor. Rapid cooling soon after death reduces the effect almost to zero. Hence, the extra tenderisation cannot be due to muscle damage during ES. Histological examination shows that stimulated muscles have longer sarcomeres than the controls; they do not exhibit damage. However, with slow cooling, irregular bands of denatured sarcoplasmic protein are deposited within the fibres of stimulated muscles, similar to those found in PSE pig muscles. There is also some shortening of sarcomeres in the region of the bands. The protein is deposited on the myofibrillar surfaces. In spite of the PSE-like appearance, there is no significant increase in drip from the stimulated muscles at 48 h after death.     

EFFECT OF ELECTRICAL STIMULATION ON CARCASSES FROM STRESSED AND UNSTRESSED STEERS

Twelve steers of known breeding and management history were assigned to two antemortem treatment groups (stress or no stress). After evisceration and splitting, sides in each group were randomly assigned to two electrical stimulation treatments (no stimulation or stimulated intermittently with one amp current for 2 min). ES did not affect any of the carcass characteristics in the stressed group except lean and overall maturity. Although ES did not affect lean color in stressed carcasses, they were darker in color than unstressed carcasses. The unstressed stimulated carcasses were more tender and contained less panel detectable connective tissue than the unstressed-unstimulated carcasses. ES did not improve tenderness of stressed carcasses. Thus, it appears that ES will be effective only on carcasses that have not severely depleted their stores of muscle glycogen.     

Structural Changes in Beef Longissimus dorsi Induced by Postmortem Low Voltage Electrical Stimulation

Advanced myofibrillar degeneration with changes of I-bands and Z-discs was noted in electrically stimulated samples as early as the day of slaughter. Occurrence of the proteolytic changes observed paralleled rate of depletion of muscle energy stores during electrical stimulation and subsequent improvements in tenderness. Localized supercontractions have been implicated as one cause of improved tenderness of electrically stimulated bovine carcasses, as has enhancement of autolytic proteolysis. Contracture bands occurred in both nonstimulated and electrically stimulated samples in the early postmortem period. Contracture bands are a common artefact in improperly prepared tissue preparations, therefore they are not a specific effect of electrical stimulation. Improved tenderness of ES samples could more easily be explained by an accelerated autolytic proteolysis.     

ENZYMIC LIPID PEROXIDATION IN BEEF MUSCLE MICROSOMES AS AFFECTED BY ELECTRICAL STIMULATION AND POSTMORTEM MUSCLE EXCISION TIME

Left sides of 10 beef carcasses were electrically stimulated at I h postmortem; right sides were not stimulated. Longissimus dorsi (LD) and trapezius (TP) muscles were removed from stimulated and nonstimulated sides after 1.5 and 24 h of chilling (0–2°C). Microsomal lipid peroxidizing activity (the amount of malonalde-hyde produced per mg of microsomal protein) was not affected by electrical stimulation, was higher for muscles removed after 24 h chilling than for those removed after 1.5 h chilling, and was higher for TP than for LD muscles. Differences observed in lipid peroxidizing activity could not be explained readily by microsomal fatty acid compositions.     

Electrical stimulation and ageing of carcasses from red,fallow and New Zealand wapiti-type male deer

Farmed male red deer (15 and 26 months), fallow deer (16 months) and New Zealand wapiti-type deer (26 months) slaughtered in an experimental abattoir were used to determine the influence of processing practices on tenderness and pH. Carcasses were electrically stimulated (low voltage) through the leg or anus immediately after bleeding and compared with non-stimulated carcasses. Portions of loin and leg from all carcasses were subjected to a range of conditioning and ageing treatments before tenderness assessment was done with a Warner-Bratzler shear press. Tenderness was measured in some samples after a freeze and slow thaw cycle. Electrical stimulation in all deer types had a highly significant effect in improving tenderness and the effects were most dramatic when carcasses were placed in a chiller 2 h after slaughter. Conditioning and ageing regimes (maximum 96 h post-slaughter) further improved tenderness, particularly in non-stimulated carcasses. Processing effects were more marked in loin pieces; these cooled more rapidly than leg. The process of freezing and slow thawing (20 h at 4°C in loins and 24–96 h at 0°C for legs) improved tenderness by 10–40% when compared with unfrozen pieces. There were highly significant reductions in the pH values of legs due to stimulation as measured during the early stages after slaughter. A decline in pH with time post-slaughter was also evident. The results were consistent with the post-slaughter pH profile of loins. Electrical stimulation, via either the leg or the anus, gave similar results. An exception was the loin pH measurement from anally stimulated wapiti stags which appeared higher than their leg-stimulated counterparts. Where data were collected from both sides of leg-stimulated carcasses, pH values were around 0.13 units lower for the stimulated shackled leg than for the free leg. Leg temperatures rose to over 40°C at 0.5 h post-slaughter in all deer. Temperatures were generally similar between stimulated and non-stimulated carcasses for both loin and leg data.