The effects of pre-slaughter pig management from the farm to the processing plant on pork quality.

Two experiments (Exp.1, n=80; Exp.2, n=144) were conducted to determine the effects of pre-slaughter pig management on pork quality by monitoring blood lactate concentration ([LAC]) during marketing. [LAC] was measured at: (1) baseline at farm, (2) post-loading on truck, (3) pre-unloading after transport, (4) post-unloading at plant, (5) post-lairage, (6) post-movement to stun, and (7) exsanguination. Pearson correlations were used to determine relationships between [LAC] and meat quality. Higher [LAC] post-loading or a greater change in [LAC] during loading resulted in increased 24h pH (P=0.002, P=0.0006, Exp.1; P=0.0001, P=0.01, Exp.2, respectively), decreased L* (P=0.03, P=0.04; P=0.001, P=0.01) and decreased drip loss (P=0.02, P=0.12; P=0.002, P=0.01). Even though improved handling during loading is important to animal well-being, it will not necessarily translate into improved pork quality.

Persistence of blood changes associated with alteration of the dietary electrolyte balance in commercial pigs after feed withdrawal, transportation, and lairage, and the effects on performance and carcass quality.

Increasing dietary electrolyte balance (dEB) has previously been shown to reduce the incidence of nonambulatory and noninjured swine, improve meat quality, and reduce the incidence of gastric ulcers. The objective of this study was to evaluate the effect of dEB under commercial conditions. Due to the variability in feed withdrawal, transport, and lairage conditions in the swine industry, it was necessary to determine first the persistence of blood changes during the marketing process after alteration of dEB. Sixteen pens of 8 crossbred barrows were assigned to a low (121 mEq/kg) or high (375 mEq/kg) dEB diet, calculated as Na(+) + K(+) - Cl(-), to determine the persistence of blood changes associated with the alteration of dEB. Diets were formulated to meet or exceed NRC (1998) requirements for energy, protein, vitamins, and minerals. Dietary treatments were provided for ad libitum intake for 3 d before slaughter. Before transport, animals were fasted in the barn for approximately 10 h. After fasting, animals were shipped to the packing plant, rested for 8 h, and subsequently slaughtered. Initial and final BW of the animals were obtained. Blood was sampled at baseline (2 d before administration of diets), before feed withdrawal (0 h), after feed withdrawal (10 h), and at exsanguination (20 h). Consumption of the high dEB diet for 3 d resulted in an increase in blood TCO(2) (P = 0.001), HCO(3)(-) (P = 0.001), and base excess (P = 0.0003) and a decrease in Cl(-) (P = 0.0002) and anion gap (P = 0.01). These differences, however, were not maintained for any of the blood components after the 10-h feed withdrawal (P > 0.22). Increasing dEB had no adverse effects (P > 0.18) on growth performance, meat quality, or carcass yield and did not decrease pars esophageal ulcer scores. This study demonstrated that the effect of dEB on blood components was not maintained after a 10-h feed withdrawal. Therefore, it is likely that the ability of the animal to withstand any increased metabolic acid load associated with the stress of transport was lost after feed withdrawal. Further research is needed to determine the effects of dEB alteration in animals that have not been fasted before shipment and using diets with a larger difference in dEB.

Use of exsanguination blood lactate to assess the quality of pre-slaughter pig handling.

The objective of these studies (Exp.1, n=76; Exp.2, n=140) was to characterize the relationship of pre-slaughter animal-handling events to exsanguination blood lactate concentration ([LAC]) in a commercial pork processing plant. Pearson correlations indicated relationships (P<0.05) between [LAC] and the number of times a pig jammed, backed up and reared (Exp.1), and [LAC] was correlated (P<0.05) with electric prod use and vocalization in response to prod use in the crowd pen, as well as jamming in the single-file chute (Exp. 2). Single degree of freedom contrasts indicated that pigs experiencing one or more events (i.e., jamming, rearing and/or backing up) while moving through a single-file chute had greater (P<0.03) [LAC] than pigs that did not experience these events in both experiments, whereas pigs prodded in the crowd pen had greater (P=0.03) [LAC] than pigs that were not prodded (Exp. 2). This study provides data demonstrating that specific pre-slaughter animal-handling events are related to post-slaughter [LAC] in a commercial setting.

The relationship between exsanguination blood lactate concentration and carcass quality in slaughter pigs.

A group of 128 cross-bred barrows were used to determine the relationship between exsanguination blood lactate concentration ([LAC]) and carcass quality following commercial marketing conditions. After 10h of feed withdrawal, pigs were loaded on a truck with a hydraulically lifted second deck and transported approximately 1h to the slaughter facility. Pigs were rested for 8h and stunned with carbon dioxide. Blood lactate concentration was measured on exsanguination blood. Fourteen pork quality measurements were obtained following normal post-mortem processing. Pearson correlations were used to determine the relationships between [LAC] and the meat quality parameters. Exsanguination blood lactate concentration ranged from 4 to 19.7 mM. Higher lactate was associated with lower 60 min pH (P=0.0004) and higher drip loss (P=0.02). These results suggest that under low-stress loading and standard marketing conditions, exsanguination [LAC] is predictive of the rate of early post-mortem metabolism.

Effects of multiple concurrent stressors on rectal temperature, blood acid-base status, and longissimus muscle glycolytic potential in market-weight pigs.

Sixty-four market-weight (130.0 +/- 0.65 kg) barrows (n = 16) and gilts (n = 48) were used in a split-plot design with a 2 x 2 x 2 factorial arrangement of treatments: 1) handling intensity (gentle vs. aggressive), 2) transport floor space (0.39 vs. 0.49 m(2)/pig), and 3) distance moved during handling (25 vs. 125 m) to determine the effects of multiple concurrent stressors on metabolic responses. For the handling intensity treatment, pigs were moved individually approximately 50 m through a handling course with either 0 (gentle) or 8 (aggressive) shocks from an electric goad. Pigs were loaded onto a trailer and transported for approximately 1 h at floor spaces of either 0.39 or 0.49 m(2)/pig. After transport, pigs were unloaded, and the distance moved treatment was applied; pigs were moved 25 or 125 m through a handling course using livestock paddles. Rectal temperature was measured, and blood samples (to measure blood acid-base status) were collected 2 h before the handling intensity treatment was applied and immediately after the distance moved treatment was applied. A LM sample to measure glycolytic potential was collected after the distance moved treatments on a subset of 32 pigs. There were handling intensity x distance moved interactions (P < 0.05) for several blood acid-base measurements. In general, there was no effect of distance moved on these traits when pigs were previously handled gently. However, when pigs were previously handled aggressively, pigs moved 125 compared with 25 m had greater (P < 0.05) blood lactate and less (P < 0.05) blood pH, bicarbonate, and base-excess. Pigs transported at 0.39 compared with 0.49 m(2)/pig had a greater (P < 0.01) increase in creatine kinase values; however, transport floor space did not affect any other measurements. Data were analyzed by the number of stressors (the aggressive handling, restricted transport floor space, and 125-m distance moved treatments) experienced by each pig (0, 1, 2, or 3). As the number of stressors experienced by the pig increased, rectal temperature, blood lactate, and LM lactate increased linearly (P

Coccidia-induced mucogenesis promotes the onset of necrotic enteritis by supporting Clostridium perfringens growth.

This study tested the hypothesis that a host mucogenic response to an intestinal coccidial infection promotes the onset of necrotic enteritis (NE). A chick NE model was used in which birds were inoculated with Eimeria acervulina and E. maxima and subsequently with Clostridium perfringens (EAM/CP). A second group of EAM/CP-infected birds was treated with the ionophore narasin (NAR/EAM/CP). These groups were compared to birds that were either non-infected (NIF), or infected only with E. acervulina and E. maxima (EAM), or C. perfringens (CP). The impact of intestinal coccidial infection and anti-coccidial treatment on host immune responses and microbial community structure were evaluated with histochemical-, cultivation- and molecular-based techniques. Barrier function was compromised in EAM/CP-infected birds as indicated by elevated CFUs for anaerobic bacteria and C. perfringens in the spleen when compared to NIF controls at day 20, with a subsequent increase in intestinal NE lesions and mortality at day 22. These results correlate positively with a host inflammatory response as evidenced by increased ileal interleukin (IL)-4, IL-10 and IFN-gamma RNA expression. Concurrent increases in chicken intestinal mucin RNA expression, and goblet cell number and theca size indicate that EAM/CP induced an intestinal mucogenic response. Correspondingly, the growth of mucolytic bacteria and C. perfringens as well as alpha toxin production was greatest in EAM/CP-infected birds. The ionophore narasin, which directly eliminates coccidia, reduced goblet cell theca size, IL-10 and IFN-gamma expression, the growth of mucolytic bacteria including C. perfringens, coccidial and NE lesions and mortality in birds that were co-infected with coccidia and C. perfringens. Collectively the data support the hypothesis that coccidial infection induces a host mucogenic response providing a growth advantage to C. perfringens, the causative agent of NE.

Effects of halothane sensitivity on mobility status and blood metabolites of HAL-1843-normal pigs after rigorous handling.

The objective of this study was to determine if HAL-1843-normal pigs that respond abnormally to halothane anesthesia were more likely to become nonambulatory (NA) when subjected to rigorous handling than pigs that exhibit a normal response to halothane. After a 1,100-km transport, pigs exhibiting low (HS-L; n = 33), intermediate (HS-I; n = 10), and high (HS-H; n = 47) sensitivity to halothane were moved through a 36.6-m long aisle that was 2.1 m wide at each end and 0.6 m wide in the middle 18.3 m. Ten groups of 8 pigs were briskly moved down the aisle and back 4 times, receiving a minimum of 1 electrical prod per pass (8 prods/pig). Before testing, rectal temperature was measured, open-mouth breathing and skin discoloration were visually evaluated, and a blood sample was collected from each pig. After the test, the pigs were returned to their pens, and the same measurements were taken immediately posttest and 1 h posttest (no blood at 1 h posttest). Pigs that were HS-H were more prone to becoming NA compared with HS-L pigs (P < 0.02). Regardless of halothane status, a greater number of pigs exhibited open-mouth breathing and skin discolorations immediately posttest than at the pretest or 1 h posttest times (P < 0.05). No differences were observed in blood metabolites between the different halothane sensitivity categories. However, pigs that became NA had elevated blood levels of creatine phosphokinase, lactate, glycerol, nonesterified fatty acids, ammonia, and urea nitrogen before testing (P < 0.05). Collectively, these data suggest HS-H pigs are more susceptible to becoming NA than HS-L. The elevated pretest blood metabolites of NA pigs suggest that they were in a hypermetabolic state that predisposed them to becoming NA.

The effects of ractopamine hydrochloride on lean carcass yields and pork quality characteristics.

One hundred eighty barrows were evaluated to determine the effects of ractopamine hydrochloride (RAC) on lean carcass yields and pork quality. The pens were blocked by weight (six pens per block) with starting block weights of 69.0, 70.7, 73.8, 76.6, 78.4, and 84.3 kg. Pens within a block were assigned randomly to one of three RAC treatments so each treatment in a block was replicated twice. Treatments (as-fed basis) included control diet, 10 ppm of RAC added (R10), and 20 ppm of RAC added (R20) and ranged from 25 to 41 d depending on block. Pigs were slaughtered by blocks when block average live weights were 109 kg. Gain and feed efficiency were improved (P < 0.05) with increasing dietary concentrations of RAC, but feed intake did not differ (P > 0.05). Dressing percentage was higher (P < 0.05) for RAC-treated pigs. Subjective color, firmness, marbling scores, and Minolta L* reflection of the LM were not different (P > 0.05) among treatments. Carcass weights were heavier (P < 0.05) for pigs treated with RAC compared with control pigs and were higher for R20 than for R10. The RAC-fed pigs had greater (P < 0.05) yields (actual and percentage of HCW) of the following Institutional Meat Purchase Specification (IMPS) cuts than control pigs: trimmed, boneless ham (IMPS-402C and IMPS-402G), loin (IMPS-414), sirloin, and Boston butt (IMPS-406A). Pigs treated with RAC had a greater (P < 0.05) percentage of fat-free lean trimmings (IMPS-418) than did control pigs. Pigs treated with the R20 concentration had increased (P < 0.05) water-holding capacity compared with control pigs. Purge loss decreased linearly (P < 0.05) with increasing RAC compared with control for 14-d aged, non-enhanced loins. Warner-Bratzler shear (WBS) force values measured for nonenhanced chops were greater for RAC-treated pigs than for control pigs with a low dose response (P = 0.001). Enhanced chop (salt and phosphate injection) WBS values did not differ (P > 0.05) among dietary treatments. Trained sensory evaluation panel results for tenderness decreased in a low-dose plateau response fashion for nonenhanced chops (P = 0.004). Tenderness of enhanced chops decreased linearly (P = 0.04) with increasing RAC concentrations. No differences (P > 0.05) were found in juiciness or flavor of enhanced or nonenhanced chops. Feeding RAC to late-finishing swine resulted in faster growing, more efficient animals with increased boneless subprimal yields, and it had little effect on pork juiciness and flavor.

The effect of dietary ractopamine concentration and duration of feeding on growth performance, carcass characteristics, and meat quality of finishing pigs.

A total of 400 barrows from Dekalb EB and 83 terminal sires mated to 43 and 45 maternal lines were used to evaluate the effects of dietary ractopamine (RAC; Paylean, Elanco Animal Health, Greenfield, IN) concentrations (0, 5, 10, or 20 ppm; as-fed basis) and feeding durations (6 to 34 d) on growth, efficiency, carcass, and meat quality characteristics of finishing pigs. Barrows were weighed and sorted into five weight blocks, each block consisting of 16 pens (five pigs per pen). Weight blocks were allocated to feeding duration treatments and assigned consecutively by weight from lightest to heaviest to represent 34, 27, 20, 13, and 6 d on test, respectively. The lightest and heaviest blocks averaged 79.8 and 103.8 kg, respectively, at the start of the test. Within a weight block, pens (four per treatment) were randomly assigned to one of four dietary concentrations of RAC in a basal diet containing 18.5% CP and 1.13% lysine. The experiment-wide target slaughter weight was 109 kg, and pigs and feeders were weighed weekly. Weight blocks (80 barrows per block) were slaughtered at a commercial packing plant after 6, 13, 20, 27, or 34 d on test. Overall, RAC supplementation improved (P < 0.05) ADG; however, ADG was not different (P > 0.08) from controls for pigs fed 5, 10, and 20 ppm RAC for 27, 34, and 6 d, respectively. During each feeding period, RAC-fed pigs had improved (P < 0.05) G:F, and, after 20, 27, and 34 d on test, pigs fed 20 ppm RAC had greater (P < 0.05) G:F compared with those fed 0 or 5 ppm RAC. Hot carcass weight was increased (P < 0.05) by RAC feeding after 13 and 27 d of feeding, and by feeding 10 and 20 ppm RAC after 20 d of feeding. After 34 d, pigs fed 20 ppm RAC had heavier (P < 0.05) hot carcass weight than pigs fed 10 ppm RAC. Fat-free lean estimates and the 10th-rib LM area were increased (P < 0.05) by feeding 10 and 20 ppm RAC after 27 d, and by feeding 20 ppm RAC after 34 d compared with controls. Japanese and American color scores, as well as L*, a*, and b* values of the LM, were not affected (P > 0.11) by 5 and 10 ppm RAC compared with controls during each feeding period. Visual marbling score for the LM was decreased (P < 0.05) when RAC was fed at 10 and 20 ppm compared with 0 ppm RAC when fed for 34 d. Dietary RAC improved growth performance at all feeding durations, whereas carcass composition was improved at longer feeding durations. In addition, 5 and 10 ppm RAC did not affect objective and subjective measures of pork quality.

Molecular ecological analysis of porcine ileal microbiota responses to antimicrobial growth promoters.

Cultivation-independent microbial molecular ecology approaches were used to examine the effects of antibiotic growth promoters on the pig ileal microbiota. Five-week-old barrows were fitted with a simple T-cannula at the distal ileum. Three diets meeting or exceeding the minimum nutrient requirements were fed for 5 wk and supplemented as follows: 1) negative control (no antibiotic; n = 5), 2) continuous tylosin administration (n = 5), and 3) an antibiotic rotation sequence (wk 1, chlorotetracycline sulfathiazole penicillin; wk 2, bacitracin and roxarsone; wk 3, lincomycin; wk 4, carbadox; wk 5, virginiamycin; n = 5). Ileal luminal contents were collected for DNA isolation at the end of each of the 5 wk of the testing period. The V3 region of 16S rDNA was amplified by PCR and analyzed via denaturing gradient gel electrophoresis (DGGE) and quantitative polymerase chain reaction (qPCR). Resulting PCR-DGGE band numbers (bacterial species) were counted, and the banding patterns analyzed by calculating Sorenson's pairwise similarity coefficients (C(S)), an index measuring bacterial species in common among samples. Band numbers and total bacterial DNA concentrations decreased (P < 0.05) temporally in antibiotic-treated pigs compared with controls. Comparisons between treatments yielded low intertreatment C(S) indices, indicating treatment-dependent alterations in banding patterns, whereas intratreatment comparisons revealed increased homogeneity in antibiotic-treated vs. control pigs. Sequence analysis of treatment-specific bands identified three Lactobacillus, one Streptococcus, and one Bacillus species that were diminished with antibiotic rotation treatment, whereas tylosin selected for the presence of L. gasseri. Lactobacillus-specific qPCR was performed and analyzed as a percentage of total bacteria to further evaluate the effects of antibiotic administration on this genus. Total bacteria were decreased (P < 0.05) by tylosin and rotation treatments, whereas the percentage of lactobacilli increased (P < 0.05) by d 14 and through d 28 in tylosin-treated pigs. The decrease in total bacteria by antibiotics may reduce host-related intestinal or immune responses, which would divert energy that could otherwise be used for growth. Conversely, the ability of tylosin to improve animal growth may relate to its apparent selection for lactobacilli, commensals known to competitively exclude potentially pathogenic species from colonizing the intestine.

Effects of tylosin on bacterial mucolysis, Clostridium perfringens colonization, and intestinal barrier function in a chick model of necrotic enteritis.

Necrotic enteritis (NE) is a worldwide poultry disease caused by the alpha toxin-producing bacterium Clostridium perfringens. Disease risk factors include concurrent coccidial infection and the dietary use of cereal grains high in nonstarch polysaccharides (NSP), such as wheat, barley, rye, and oats. Outbreaks of NE can be prevented or treated by the use of in-feed antibiotics. However, the current debate regarding the prophylactic use of antibiotics in animal diets necessitates a better understanding of factors that influence intestinal colonization by C. perfringens as well as the pathophysiological consequences of its growth. We report a study with a chick model of NE, which used molecular (16S rRNA gene [16S rDNA]) and culture-based microbiological techniques to investigate the impact of the macrolide antibiotic tylosin phosphate (100 ppm) and a dietary NSP (pectin) on the community structure of the small intestinal microbiota relative to colonization by C. perfringens. The effects of tylosin and pectin on mucolytic activity of the microbiota and C. perfringens colonization and their relationship to pathological indices of NE were of particular interest. The data demonstrate that tylosin reduced the percentage of mucolytic bacteria in general and the concentration of C. perfringens in particular, and these responses correlated in a temporal fashion with a reduction in the occurrence of NE lesions and an improvement in barrier function. The presence of pectin did not significantly affect the variables measured. Thus, it appears that tylosin can control NE through its modulation of C. perfringens colonization and the mucolytic activity of the intestinal microbiota.

Antibiotics as growth promotants: mode of action.

Recent concerns about the use of growth-promoting antibiotics in pig diets have renewed interest in the immunologic and growth-regulating functions of the gastrointestinal (GI) tract. The numerically dense and metabolically active microbiota ofthe pig GI tract represents a key focal point for such questions. The intestinal microbiota is viewed typically as a beneficial entity for the host. Intestinal bacteria provide both nutritional and defensive functions for their host. However, the host animal invests substantially in defensive efforts to first sequester gut microbes away from the epithelial surface, and second to quickly mount immune responses against those organisms that breach epithelial defenses. The impact of host responses to gut bacteria and their metabolic activities require special consideration when viewed in the context of pig production in which efficiency of animal growth is a primary objective. Here, we summarize the working hypothesis that antibiotics improve the efficiency of animal growth via their inhibition of the normal microbiota, leading to increased nutrient utilization and a reduction in the maintenance costs ofthe GI system. In addition, novel molecular ecology techniques are described that can serve as tools to uncover the relationship between intestinal microbiology and growth efficiency.

Paylean alters myosin heavy chain isoform content in pig muscle.

Feeding beta-adrenergic agonists promotes muscle growth. Early histological techniques failed to show precisely how feeding ractopamine-HCl (Paylean) alters muscle growth in pigs. To understand these effects, an indirect enzyme-linked immunosorbent assay (ELISA) was used to determine the abundance of each adult skeletal muscle myosin heavy chain isoform, one means of assigning muscle fiber type, in fast and slow muscles of pigs fed Paylean. Sixty growing pigs (-85 kg) were randomly assigned to three Paylean doses (0, 20, or 60 ppm). At 3, 7, 14, 28, and 42 d of treatment, four pigs per dose were harvested and white (WST) and red (RST) semitendinosus and longissimus (LM) muscles were removed and processed, and myosin heavy chain was quantified by ELISA. Feeding Paylean enhanced (P < 0.05) pigs' average daily gain. Muscle myosin heavy chain (slow, 2A, 2AX, and 2B) composition differed (P < 0.05) across muscles. Compared with LM, RST contained approximately five times more (P < 0.0001) slow and type 2A myosin heavy chain and three times more 2AX myosin heavy chain but nearly undetectable amounts of 2B myosin heavy chain. Myosin heavy chain composition of the WST closely resembled that of the LM (i.e., greater 2AX and 2B and less slow and 2A). After 42d of 60 ppm Paylean, the amount of slow, 2A, and 2AX myosin heavy chain decreased (P < 0.05) across the three muscles whereas the amount of 2B myosin heavy chain increased (P < 0.05). In contrast, relative amounts of 2A and 2AX myosin heavy chain increased (P < 0.05) in muscle of control pigs at 42d. Changes associated with the 20-ppm dose were intermediate to and different from (P < 0.05) control and 60 ppm treatments. Correlations (P < 0.05) among various myosin heavy chain within muscles suggest that slow, type 2A, and 2X decrease with increases in 2B myosin heavy chain. These data show that administration of Paylean affects myosin heavy chain isoform composition in a time- and dose-dependent manner and provides a mechanism of action for Paylean altering animal growth.

Expression of Acidothermus cellulolyticus endoglucanase E1 in transgenic tobacco: biochemical characteristics and physiological effects.

The expression of the Acidothermus cellulolyticus endoglucanase E1 gene in transgenic tobacco (Nicotiana tabacum) was examined in this study, where E1 coding sequence was transcribed under the control of a leaf specific Rubisco small subunit promoter (tomato RbcS-3C). Targeting the E1 protein to the chloroplast was established using a chloroplast transit peptide of Rubisco small subunit protein (tomato RbcS-2A) and confirmed by immunocytochemistry. The E1 produced in transgenic tobacco plants was found to be biologically active, and to accumulate in leaves at levels of up to 1.35% of total soluble protein. Optimum temperature and pH for E1 enzyme activity in leaf extracts were 81 degrees C and 5.25, respectively. E1 activity remained constant on a gram fresh leaf weight basis, but dramatically increased on a total leaf soluble protein basis as leaves aged, or when leaf discs were dehydrated. E1 protein in old leaves, or after 5 h dehydration, was partially degraded although E1 activity remained constant. Transgenic plants exhibited normal growth and developmental characteristics with photosynthetic rates similar to those of untransformed SR1 tobacco plants. Results from these biochemical and physiological analyses suggest that the chloroplast is a suitable cellular compartment for accumulation of the hydrolytic E1 enzyme.

Pseudotyping of glycoprotein D-deficient herpes simplex virus type 1 with vesicular stomatitis virus glycoprotein G enables mutant virus attachment and entry.

The use of herpes simplex virus (HSV) vectors for in vivo gene therapy will require the targeting of vector infection to specific cell types in certain in vivo applications. Because HSV glycoprotein D (gD) imparts a broad host range for viral infection through recognition of ubiquitous host cell receptors, vector targeting will require the manipulation of gD to provide new cell recognition specificities in a manner designed to preserve gD's essential role in virus entry. In this study, we have determined whether an entry-incompetent HSV mutant with deletions of all Us glycoproteins, including gD, can be complemented by a foreign attachment/entry protein with a different receptor-binding specificity, the vesicular stomatitis virus glycoprotein G (VSV-G). The results showed that transiently expressed VSV-G was incorporated into gD-deficient HSV envelopes and that the resulting pseudotyped virus formed plaques on gD-expressing VD60 cells, albeit at a 50-fold-reduced level compared to that of wild-type gD. This reduction may be related to differences in the entry pathways used by VSV and HSV or to the observed lower rate of incorporation of VSV-G into virus envelopes than that of gD. The rate of VSV-G incorporation was greatly improved by using recombinant molecules in which the transmembrane domain of HSV glycoprotein B or D was substituted for that of VSV-G, but these recombinant molecules failed to promote virus entry. These results show that foreign glycoproteins can be incorporated into the HSV envelope during replication and that gD can be dispensed with on the condition that a suitable attachment/entry function is provided.

Emerging cytopathic and antigenic simian immunodeficiency virus variants influence AIDS progression.

Genetic variants of human and simian immunodeficiency virus (HIV and SIV) that evolve during the course of infection and progression to AIDS are phenotypically and antigenically distinct from their progenitor viruses present at early stages of infection. However, it has been unclear how these late variants, which are typically T-cell tropic, cytopathic and resistant to neutralizing antibodies, influence the development of clinical AIDS. To address this, we infected macaques with cloned SIVs representing prototype variants from early-, intermediate- and late-stage infection having biological characteristics typical of viruses found at similar stages of HIV infection in humans. These studies demonstrate that sequential, phenotypic and antigenic variants represent viruses that have become increasingly fit for replication in the host, and our data support the hypothesis that emerging variants have increased pathogenicity and drive disease progression in SIV and HIV infection.

Recombinant herpes simplex virus type 1 engineered for targeted binding to erythropoietin receptor-bearing cells.

The utility of recombinant herpes simplex virus type 1 (HSV-1) vectors may be expanded by manipulation of the virus envelope to achieve cell-specific gene delivery. To this end, an HSV-1 mutant virus deleted for glycoprotein C (gC) and the heparan sulfate binding domain of gB (KgBpK-gC-) was engineered to encode different chimeric proteins composed of N-terminally truncated forms of gC and the full-length erythropoietin hormone (EPO). Biochemical analyses demonstrated that one gC-EPO chimeric molecule (gCEPO2) was posttranslationally processed, incorporated into recombinant HSV-1 virus (KgBpK-gCEPO2), and neutralized with antibodies directed against gC or EPO in a complement-dependent manner. Moreover, KgBpK-gCEPO2 recombinant virus was specifically retained on a soluble EPO receptor column, was neutralized by soluble EPO receptor, and stimulated proliferation of FD-EPO cells, an EPO growth-dependent cell line. FD-EPO cells were nevertheless refractory to productive infection by both wild-type HSV-1 and recombinant KgBpK-gCEPO2 virus. Transmission electron microscopy of FD-EPO cells infected with KgBpK-gCEPO2 showed virus endocytosis leading to aborted infection. Despite the lack of productive infection, these data provide the first evidence of targeted HSV-1 binding to a non-HSV-1 cell surface receptor.

Heparan sulfate proteoglycan binding by herpes simplex virus type 1 glycoproteins B and C, which differ in their contributions to virus attachment, penetration, and cell-to-cell spread.

Herpes simplex virus type 1 (HSV-1) mutants defective for envelope glycoprotein C (gC) and gB are highly impaired in the ability to attach to cell surface heparan sulfate (HS) moieties of proteoglycans, the initial virus receptor. Here we report studies aimed at defining the HS binding element of HSV-1 (strain KOS) gB and determining whether this structure is functionally independent of gB's role in extracellular virus penetration or intercellular virus spread. A mutant form of gB deleted for a putative HS binding lysine-rich (pK) sequence (residues 68 to 76) was transiently expressed in Vero cells and shown to be processed normally, leading to exposure on the cell surface. Solubilized gBpK- also had substantially lower affinity for heparin-acrylic beads than did wild-type gB, confirming that the HS binding domain had been inactivated. The gBpK- gene was used to rescue a KOS gB null mutant virus to produce the replication-competent mutant KgBpK-. Compared with wild-type virus, KgBpK- showed reduced binding to mouse L cells (ca. 20%), while a gC null mutant virus in which the gC coding sequence was replaced by the lacZ gene (KCZ) was substantially more impaired (ca. 65%-reduced binding), indicating that the contribution of gC to HS binding was greater than that of gB. The effect of combining both mutations into a single virus (KgBpK-gC-) was additive (ca. 80%-reduced binding to HS) and displayed a binding activity similar to that observed for KOS virus attachment to sog9 cells, a glycosaminoglycan-deficient L-cell line. Cell-adsorbed individual and double HS mutant viruses exhibited a lower rate of virus entry following attachment, suggesting that HS binding plays a role in the process of virus penetration. Moreover, the KgBpK- mutant virus produced small plaques on Vero cells in the presence of neutralizing antibody where plaque formation depended on cell-to-cell virus spread. These studies permitted the following conclusions: (i) the pK sequence is not essential for gB processing or function in virus infection, (ii) the lysine-rich sequence of gB is responsible for HS binding, and (iii) binding to HS is cooperatively linked to the process of efficient virus entry and lateral spread but is not absolutely required for virus infectivity.

Herpes simplex virus type 1 glycoprotein B requires a cysteine residue at position 633 for folding, processing, and incorporation into mature infectious virus particles.

Herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) resides in the virus envelope in an oligomeric form and plays an essential role in virus entry into susceptible host cells. The oligomerizing domain is a movable element consisting of amino acids 626 to 653 in the gB external domain. This domain contains a single cysteine residue at position 633 (Cys-633) that is predicted to form an intramolecular disulfide bridge with Cys-596. In this study, we examined gB oligomerization, processing, and incorporation into mature virus during infection by two mutant viruses in which either the gB Cys-633 [KgB(C633S)] or both Cys-633 and Cys-596 [KgB(C596S/C633S)] residues were mutated to serine. The result of immunofluorescence studies and analyses of released virus particles showed that the mutant gB molecules were not transported to the cell surface or incorporated into mature virus envelopes and thus infectious virus was not produced. Immunoprecipitation studies revealed that the mutant gB molecules were in an oligomeric configuration and that these mutants produced hetero-oligomers with a truncated form of gB consisting of residues 1 to 43 and 595 to 904, the latter containing the oligomerization domain. Pulse-chase experiments in combination with endoglycosidase H treatment determined that the mutant molecules were improperly processed, having been retained in the endoplasmic reticulum (ER). Coimmunoprecipitation experiments revealed that the cysteine mutations resulted in gB misfolding and retention by the molecular chaperones calnexin, calreticulin, and Grp78 in the ER. The altered conformation of the gB mutant glycoproteins was directly detected by a reduction in monoclonal antibody recognition of two previously defined distinct antigenic sites located within residues 381 to 441 and 595 to 737. The misfolded molecules were not transported to the cell surface as hetero-oligomers with wild-type gB, suggesting that the conformational change could not be corrected by intermolecular interactions with the wild-type molecule. Together, these experiments confirmed that a disulfide bridge involving Cys-633 and Cys-596 is not essential for oligomerization but rather is required for proper folding and maintenance of a gB domain essential to complete posttranslational modification, transport, and incorporation into mature virus particles.

The effect of narasin on apparent nitrogen digestibility and large intestine volatile fatty acid concentrations in finishing swine.

The effect of narasin on apparent nitrogen and dry matter digestibilities and large intestine VFA concentrations in finishing swine was investigated. The study used 21 crossbred barrows averaging 72 kg. Seven blocks were formed on the basis of pretreatment dry matter digestibility, and barrows were randomly assigned to three treatments in each block. Treatments consisted of a control (C) and narasin (N15 and N30) applied at 15 and 30 ppm, respectively. Fecal and urine samples were collected. Upon the completion of the digestibility work, intestinal samples were taken from three locations, and VFA concentrations for each animal were measured. Weight gains for the N15 and N30 treatments were increased 3.0 and 6.0% (not significant), respectively, over control. Fecal nitrogen was decreased (P < .05) in the narasin-fed barrows, and apparent nitrogen digestibility was increased (P < .05). Neither nitrogen retention nor urinary nitrogen excretion was altered (P > .05) due to narasin. There were no increases (P > .05) in apparent dry matter digestibility due to narasin. Analysis of pooled colon samples showed an increase (P < .05) in the concentration of propionic acid in relation to acetic and butyric in the narasin-fed barrows. Butyric acid was reduced (P < .05) in the transverse colon of narasin-fed barrows. In summary, narasin administration to finishing barrows resulted in improved apparent nitrogen digestibility, thus decreasing fecal nitrogen, and increased relative concentrations of propionic acid in the large intestine.