Cytotoxicity against autologous, allogeneic, and xenogeneic tumor targets by human recombinant interleukin-2-activated lymphocytes from healthy dogs and dogs with lung tumors

Before dogs with lung tumors were treated by adoptive immunotherapy, the ability of canine blood lymphocytes (PBL) from the peripheral circulation to differentiate in vitro in the presence of human recombinant interleukin-2 (rIL-2) and become tumoricidal was investigated. The PBL from healthy dogs (n = 6) and dogs with lung tumors (n = 5) were grown in culture medium alone, in the presence of rIL-2 to generate lymphokine-activated killer (LAK) cells, or with phytohemagglutinin (PHA) and rIL-2 to generate autologous-stimulated lymphocytes (ASL). After 4 days, cytotoxicity by the ASL, LAK, and PBL was determined in a 4-hour 51chromium-release assay. Target cells in the assay were short-term cultured enzyme digests of autologous (self), allogeneic (genetically different) primary tumors, and Raji, the xenogeneic human lymphoma cell line. The PBL cultured without rIL-2 were not cytotoxic against any tumor. However, when a dog's PBL were activated in vitro, they killed the dog's own tumor, ASL more effectively than LAK cells. Pulmonary adenocarcinomas and an osteosarcoma metastasis to lung were among the autologous tumors assayed. Against an allogeneic canine osteosarcoma, ASL generated from healthy dogs were significantly more cytolytic than LAK from healthy dogs, or than ASL generated from tumor-bearing dogs. Cytotoxicity was greater against allogeneic tumor than against Raji. Lectin-dependent cellular cytotoxicity, tested by including PHA in the assay medium with lymphocytes and Raji cells, by ASL and LAK was greater than cytotoxicity of Raji without PHA. Because ASL were more cytolytic than LAK against all targets in vitro, they may be more beneficial than LAK for immunotherapy of canine tumors.     

Optimization of conditions for in vitro production of radical oxygen species and expression of tissue factor by canine mononuclear cells and granulocytes for use in high-throughput assays

The purpose of this study was to optimize conditions for high throughput measurement of radical oxygen species (ROS) production and expression of tissue factor, also termed procoagulant activity, by canine leukocytes. Granulocytes and mononuclear cells were separated by density gradient centrifugation from peripheral blood collected on several occasions from three healthy large breed dogs. To determine optimal conditions for ROS production, granulocytes were incubated for 1 or 3h in PBG (PBS containing 0.5% BSA and 5mM glucose) or RPMI containing 10% fetal bovine serum (FBS); lipopolysaccharide (LPS), zymosan, peptidoglycan (PGN) and phorbol myristate acetate (PMA) were used as stimuli. ROS was assessed by conversion of the nonfluorescent dye dihydrorhodamine 123 to fluorescent rhodamine 123 by radical species released into the media. To identify optimal conditions for expression of tissue factor, mononuclear cells were incubated for 5h in RPMI containing different concentrations of heat-inactivated FBS (HI-FBS), and LPS, zymosan, PGN or PMA as stimuli. Expression of tissue factor was determined using a one-stage recalcification assay performed in an automated nephelometric coagulation analyzer. Neither LPS nor zymosan increased ROS production by granulocytes incubated in PBG media. In contrast, granulocytes incubated in RPMI had dose-dependent increases in ROS production in response to zymosan and PGN. ROS production was significantly increased by incubation with concentrations of LPS of 0.01microg/ml or greater, and by zymosan concentrations of 0.1microg/ml or greater. ROS production in response to incubation with PMA was significantly increased starting at 10(-7)M, and was significantly greater for cells incubated in RPMI than cells incubated in PBG. LPS-, zymosan- and PGN-stimulated procoagulant activity increased in a dose-dependent manner, whereas PMA-stimulated procoagulant activity peaked at 10(-7)M. Increasing concentrations of HI-FBS significantly increased LPS-, zymosan- and PGN-induced procoagulant activity of mononuclear cells. Results obtained in this study indicate production of ROS by canine granulocytes is optimal when these cells are incubated for 3h in RPMI with LPS (0.1microg/ml), zymosan (10 microg/ml), PGN (10 microg/ml), and PMA (10(-7)M). Furthermore, canine mononuclear cells express procoagulant activity in response to LPS, zymosan, PGN, and PMA, and responses to LPS, zymosan and PGN are enhanced by the addition of HI-FBS. These findings suggest that HI-FBS retains important serum proteins that facilitate interactions between each of these bacterial or yeast derived products and the mononuclear cells. Consequently, future studies regarding the regulation of procoagulant activity by canine mononuclear cells should be performed in the presence of HI-FBS. Both assays utilized in this study allow high throughput of samples, and therefore are appropriate choices for rapid screening of conditions and/or therapeutic interventions affecting the canine inflammatory system.     

Difference in rates of net portal absorption between crystalline and protein-bound lysine and threonine in growing pigs fed once daily

Net portal absorption of AA during the 6-h postprandial period was measured in eight gilts (48.5 +/- 1.6 kg BW) in a crossover design. The pigs had chronic catheters placed in the portal vein, carotid artery, and ileal vein, and were trained to consume 1.2 kg of a standard grower diet once daily. Blood samples were taken every 30 min for 4 h and then hourly until 6 h after feeding. The first set of blood samples was taken after pigs were fed a meal of the test 16% CP corn-soybean meal diet (16% CP) or the test 12% CP corn-soybean meal diet supplemented with crystalline lysine, threonine, and tryptophan (12% CP + AA) to equal the three AA levels in the 16% CP diet. Pigs were then fed the standard diet for 2 d. Following that, blood samples were again taken after the pigs were fed a meal of the test diet that was not given to them at the first sampling period. Net portal AA absorption was calculated by multiplying porto-arterial plasma AA concentration difference by portal vein plasma flow rate (PVPF), estimated by an indicator-dilution technique employing p-aminohippuric acid as the indicator infused into the ileal vein. Plasma concentrations of lysine and threonine of pigs were affected by the diet x time interaction (P < 0.01). Portal and arterial plasma lysine and threonine concentrations in pigs attained the maximal level by 1 h postprandial when the 12% CP + AA diet was fed, but reached the peak level at 2.5 h postprandial when the 16% CP diet was given. The PVPF of pigs over the 6 h postprandial was less (P < 0.01) when the 12% CP + AA diet was given than when the 16% CP diet was fed. Net portal absorptions of lysine and threonine also were affected (P < 0.05) by time x diet interaction. The peak portal absorption of both lysine and threonine in pigs appeared at 0.5 h postprandial when the 12% CP + AA diet was given, but at 2.5 h postprandial with the feeding of the 16% CP diet. The early appearance of peak portal absorption of lysine and threonine from feeding the 12% CP + AA compared with the 16% CP diet indicates that crystalline lysine and threonine are absorbed more rapidly than protein-bound lysine and threonine in pigs fed once daily.     

Effects of dietary fat on growth performance and carcass characteristics of growing-finishing pigs reared in a commercial environment.

We conducted two experiments to evaluate the effects of added choice white grease on performance and carcass merit of barrows and gilts reared under commercial conditions. Pigs were housed either 20 (Exp. 1) or 25 (Exp. 2) per pen and were provided 0.67 m2 of pen space per pig. Diets were based on corn and soybean meal and fed in a meal form. The proportion of soybean meal was increased in diets with added fat to maintain the same calorie:lysine ratio in all diets within a weight phase. In Exp. 1, 480 pigs were fed diets with 0, 2, 4, or 6% fat. Total lysine contents of the control diets were 1.21, 0.88, and 0.66% during the weight phases 36 to 59, 59 to 93, and 93 to 120 kg, respectively. Gain:feed was increased linearly (P < 0.01) due to fat addition in all weight intervals and over the total experiment. The effect of added fat on ADG was not consistent among the weight phases; a linear (P < 0.01) improvement was found from 36 to 59 kg, but no effect was found during the heavier weight phases. Over the total experiment, however, ADG was improved (P < 0.01) linearly. Carcass traits were not affected by treatment. Experiment 2 used 900 pigs to evaluate possible carryover effects on performance and carcass merit from feeding 6% fat. The experiment was divided into four phases: 25 to 45, 45 to 70, 70 to 90, and 90 to 115 kg; lysine contents of the control diets fed in each phase were 1.23, 1.05, 0.81, and 0.63%, respectively. The six treatments consisted of no added fat throughout the experiment or 6% added fat fed from 25 to 45 kg, 25 to 70 kg, 25 to 90 kg, 25 to 115 kg, or 45 to 70 and 90 to 115 kg. Carryover effects for ADG and G:F (P < 0.07) were found for the 90- to 115-kg interval and for ADFI and ME intake (P < 0.05) for the 45- to 70- and 70-to 90-kg intervals. When fat was added in the previous weight interval, ADG and G:F were improved and ADFI and ME intake were decreased in the subsequent weight interval. Pigs fed fat from 25 to 115 kg had more (P < 0.05) backfat and lower (P < 0.05) carcass leanness than pigs on the other treatments. These data suggest that fat can be added or removed from diets of growing-finishing pigs without any detrimental carryover effects. In fact, the positive carryover effect on ADG and G:F from 95 to 115 kg suggests that feeding fat from 25 to 95 kg will maximize performance over the total growing-finishing period but minimize any detrimental effects of added fat on carcass leanness.     

Soluble matrix of hen's eggshell extracts changes in vitro the rate of calcium carbonate precipitation and crystal morphology

1. Extra and intramineral eggshell matrix proteins were solubilised before and after demineralisation by sequential extractions using guanidine hydrochloride and EDTA.

2. The intramineral electrophoretic profile of SDS‐PAGE showed the presence of 80, 66, 43, 36 and 15 kDa bands with a predominance of a 17 kDa band. In the extramineral part, the major protein was the 15 kDa band.

3. The introduction of intramineral extract to a metastable solution of calcium carbonate delayed the rate of crystal growth. The delay in the rate of precipitation was elicited by a single fraction (MW 50–80 kDa), isolated by gel filtration chromatography, of eggshell extracts. Extramineral extracts had no effect.

4. Addition in vitro of intramineral eggshell extracts modified the morphology of calcite; the crystals aggregated and showed irregular surfaces.

5. These observations suggest that constituents of the eggshell matrix are involved in the control of calcite growth and crytallographic structure of the hen's eggshell.     

Analysis of the reactivity of anti-bovine CD8 monoclonal antibodies with cloned T cell lines and mouse L-cells transfected with bovine CD8

Mouse L-cells transfected with bovine CD8 and two Theileria parva-infected cloned T cell lines expressing bovine CD8 were used to screen the panel of ten monoclonal antibodies (mAbs) submitted to the workshop. Eight of the ten mAbs reacted with the transfectant and both the cloned T cell lines. However, two mAbs CC58 and BAT82A did not recognise the transfectant and only reacted with one of the T cell lines. Further biochemical studies indicated that the eight mAbs react with both homo- and heterodimeric forms of bovine CD8 whilst the two mAbs CC58 and BAT82A react with only heterodimeric forms. These data suggest that bovine DC8 is encoded by two genes as is the case in mouse and man.     

Uterine environment as a regulator of birth weight and body dimensions of newborn lambs

Pure-bred embryos were transferred within and reciprocally between large (Suffolk) and small (Cheviot) breeds of sheep to establish 4 treatment groups: SinS (Suffolk embryos in Suffolk dams), SinC (Suffolk embryos in Cheviot dams), CinS (Cheviot embryos in Suffolk dams), and CinC (Cheviot embryos in Cheviot dams). The recipient ewes carried single fetuses to term. The maternal plasma concentrations of ovine placental lactogen (oPL), progesterone, IGF-1, FFA, and glucose were measured on d 50, 90, 120, and 140 of pregnancy. Birth weight, body dimensions, and placental characteristics of lambs were recorded at birth. There was a recipient ewe breed × lamb breed × time interaction for the concentration of oPL (P = 0.03), but no such interaction was observed for progesterone (P = 0.42), IGF-1 (P = 0.57), glucose (P = 0.36), or FFA (P = 0.72). There were no differences in oPL (P = 0.28) and progesterone (P = 0.34) concentrations between SinC and SinS ewes. The concentrations of FFA on d 140 (P = 0.008), and those of glucose on d 50 (P = 0.02) and 120 (P = 0.01), were greater in SinC ewes than in SinS ewes. The ewes in CinS had less FFA concentration (P = 0.002) at all time points than CinC ewes. The concentrations of IGF-1 on d 90 were greater (P = 0.004) in CinS ewes than CinC ewes, but did not differ (P = 0.16) on d 50, 120, and 140. The concentrations of glucose on d 50 (P = 0.001), 90 (P = 0.03), and 140 (P = 0.03) were less in CinS ewes compared with CinC ewes. The birth weight of SinC lambs (5.04 ± 0.20 kg) was lighter (P = 0.001) than SinS lambs (5.94 ± 0.19 kg), and body dimensions of SinC lambs were smaller (P = 0.01) than SinS lambs. Neither birth weight nor the body dimensions of CinS lambs differed (P = 0.24) from CinC lambs. Cotyledon number was reduced (P = 0.04) in the CinS (57.5 ± 6.3) compared with the SinS group (74.2 ± 5.9), whereas mean cotyledon weight in CinS (2.42 ± 0.20 g) was greater (P = 0.02) than SinS (1.74 ± 0.21 g). It was concluded that the large genotype lambs were lighter and smaller when born to small genotype dams; however, the birth weight or body dimensions of small genotype lambs did not differ when born to large genotype dams. This study suggests that plasma oPL, progesterone, IGF-1, FFA, and glucose concentrations at different times throughout pregnancy reflect the regulatory effect of the uterine environment on the development of the fetus.     

Risk factors for metritis in Danish dairy cows

A retrospective longitudinal study of metritis was conducted in Denmark on data collected during 1993-1994. Data on herd size, breed, parity, and treatment of disease were obtained from the Danish Cattle Database. Management and production-facility data were collected using a questionnaire, conducted as a telephone interview in 1994. The study included 2144 herds from three regions in Denmark (102,060 cows). Herd-level variables included were: herd size, housing, flooring, grazing, calving measures, and calving supervision. Cow-level variables were: parity, breed, calving season and whether the cow had been treated by a veterinarian for dystocia or the diseases: retained placenta, reproductive disease, ketosis, milk fever, or dry cow mastitis.Marginal multivariable logistic-regression analyses were performed. The cow with highest odds of metritis was a first or greater than or equal to third parity cow, of large breed, that calved during November-April, in a zero-grazing herd. The cow had been treated for dystocia, retained placenta, and at least one other reproductive disease, but not for ketosis.     

Evaluation of protective immunity in pigs following oral administration of an avirulent live vaccine of Lawsonia intracellularis

OBJECTIVE: To evaluate the efficacy of an orally administered avirulent live vaccine to protect pigs against challenge exposure with virulent Lawsonia intracellularis. ANIMALS: 108 weaned 3-week-old pigs (35 in experiment 1 and 73 in experiment 2). PROCEDURE: 2 experiments were conducted. On day 0, vaccinates were orally administered vaccine via drench or in drinking water, whereas challenge-control pigs were administered cultured medium. On day 21, pigs were challenge exposed with a virulent heterologous isolate of L. intracellularis. Clinical observations, weights, seroconversion, and fecal excretion of L. intracellularis were measured until day 42. At study termination, pigs were euthanatized and examined for L. intracellularis-specific lesion development of the ileum and colon. RESULTS: Pigs receiving a single dose of vaccine were protected when challenge exposed with virulent L. intracellularis (at least 10(77) TCID50/dose). In experiment 1, vaccinates had significantly less fecal excretion (47% and 40% for days 35 and 42, respectively), compared with challenge-control pigs. In experiment 2, vaccinates had significantly less fecal excretion (50% and 58% for days 35 and 42, respectively), compared with challenge-control pigs. Significant reductions in lesion development were evident in the ileum of vaccinated pigs (70% and 56% at day 42 for experiments 1 and 2, respectively), compared with challenge-control pigs. CONCLUSIONS AND CLINICAL RELEVANCE: Oral administration by drench or via drinking water of an avirulent live vaccine against L. intracellularis resulted in substantial protection against proliferative enteropathy among vaccinates and offers a better way to reduce stress of pigs during vaccine administration.     

Evaluating processing temperature and feeding value of extruded-expelled soybean meal on nursery and finishing pig growth performance

We conducted two experiments comparing the use of extruded-expelled soybean meal (EESoy) to solvent-extracted soybean meal (SBM) in swine diets. In Exp. 1, the objective was to determine the optimal processing temperature of EESoy for nursery pig growth performance. Pigs (n = 330, 13.2 +/- 2.3 kg of BW) were fed a control diet containing SBM with added fat or one of five diets containing EESoy extruded at 143.3, 148.9, 154.4, 160.0, or 165.6 degrees C. All diets were formulated on an equal apparent digestible lysine:ME ratio. From d 0 to 20, no differences were observed (P > 0.32) in ADG or ADFI (average of 544 and 924 g/d, respectively). However, gain:feed ratio (G/F) improved (quadratic, P < 0.01, range of 0.56 to 0.60) with increasing processing temperature, with the greatest improvement at 148.9 degrees C. In Exp. 2, the objective was to determine the feeding value of EESoy relative to SBM with or without added fat for growing-finishing pigs in a commercial production facility. A total of 1,200 gilts (initially 24.5 +/- 5.1 kg of BW) was used, with 25 pigs per pen and eight replications per treatment. Dietary treatments were arranged in a 2 x 3 factorial, with two sources of soybean meal (SBM or EESoy) and three levels of added fat. Pigs were phase-fed four diets over the experimental period and added fat (choice white grease) levels were 0, 3.4, and 7% initially, with the added fat levels decreasing in the next three dietary phases. Energy levels were based such that the higher energy in EESoy (with or without added fat) was calculated to be equal to that provided by SBM with added fat. From 24.5 to 61.2 kg, pigs fed EESoy had greater (P < 0.07) G/F than those fed SBM. Increasing added fat in either EESoy- or SBM-based diets increased G/F (linear, P < 0.0003). From 61.2 to 122.5 kg, ADG and G/F were unaffected in pigs fed EESoy and/or increasing added fat (P > 0.10). For the overall growing-finishing period, ADG was unaffected (P > 0.61) by increasing energy density of the diet; however, ADFI decreased (P < 0.05) and G/F increased (P < 0.02, range of 0.37 to 0.40) as energy density increased with either EESoy or added fat. Carcass leanness was not affected by dietary treatment. These results indicate that EESoy should be extruded at 148.9 to 154.4 degrees C, and that increasing dietary energy density by using EESoy and/or added fat improves feed efficiency in finishing pigs reared in a commercial environment.