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.     

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.     

Improved milk production through PG–PL system by provision of in‐house shelter management in lactating Murrah buffaloes during winter season

Comprehensive information on the role of β‐casein and plasminogen–plasmin (PG–PL) system in milk secretion of Murrah buffaloes during winter season is lacking, although effects of cold stress can be ameliorated to an extent by altering microclimate at farm level. Hence, this study was aimed to determine the changes in productivity along with PG–PL system of milk, plasma hormones and metabolites of buffaloes during winter (December–January) season under two different management systems. Average minimum temperature and wind chill index during this season were 7.02 and 12.74 °C respectively. Buffaloes were divided in two groups of six animals each: control and treatment, where treatment group animals were placed in‐house with floor bedding of paddy straw and the control group animals in loose housing system without straw bedding. Physiological responses were recorded, and milk and blood samples were collected at weekly intervals for six‐week experimental period. Under in‐house management system, buffaloes experienced better comfort by alleviating environmental stress as their physiological responses such as respiration rate and pulse rate were significantly reduced (p < 0.01) as compared to the control, which subsequently resulted higher milk yield by 9.92% (p < 0.05). Analysis of milk samples revealed higher concentration of plasminogen (10.6 vs. 8.05 μg/ml; p < 0.01) and β‐casein (p < 0.05), and lower plasmin level (0.299 vs. 0.321 μg/ml; p < 0.05) in buffaloes under treatment group. It was also found that plasma cortisol, glucose and non‐esterified fatty acids levels were higher (p < 0.01) in control group as compared to the treatment animals by 13.6%, 8.14% and 12.6% respectively. However, milk composition, growth hormone, epinephrine and norepinephrine level in plasma were similar in both the groups. Hence, it may be concluded that provision of in‐house shelter management with floor bedding of paddy straw during winter was effective to minimize environmental stress and improved milk production through manipulation of PG–PL system in buffaloes.     

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.     

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.     

Ribotyping of Indian Isolates of <Emphasis Type="Italic">Pasteurella multocida</Emphasis> Based on 16S and 23S rRNA Genes

The applicability of ribotyping based on 16S and 23S rRNA was evaluated for molecular epidemiological studies. Forty-eight isolates of Pasteurella multocida isolated from different hosts and geographical locations and one reference isolate were ribotyped. Only four ribotypes were found. All the isolates including reference isolate from wild carnivores had the same ribotype, though they had different serotypes. The isolate from a tiger had one band in addition to the bands present in the major ribotype. The isolates from lions represented two ribotypes; of these ribotypes, one (r2) had an additional band of 3.6 kbp, which was absent in all other ribotypes. The second ribotype (r4) from a lion had one band missing (6 kbp) that was present in the other ribotypes. These isolates were further typed using ERIC-PCR and REP-PCR. With ERIC-PCR and REP-PCR, higher D values of 0.83 and 0.89 were obtained. The current study revealed that ribotyping is not a very efficient typing tool for use in molecular epidemiology for differentiation of isolates.     

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.     

Comparison of slot blot nucleic acid hybridization, immunofluorescence, and virus isolation techniques to detect bluetongue virus in blood mononuclear cells from cattle with experimentally induced infection.

A slot blot hybridization technique was applied for detection of bluetongue virus (BTV) in blood mononuclear cells (BMNC) obtained from cattle with experimentally induced infection. This technique lacked sensitivity to detect the viral nucleic acid directly in clinical specimens. When aliquots of mononuclear cells from these cattle were cultivated in vitro for 10 days to amplify virus titer, only 33.3% of the samples collected during viremia gave a positive signal in the slot blot hybridization format. By contrast, results for 34.3% of noncultured and 63.3% of cultured mononuclear cell samples collected during viremia were positive by immunofluorescence. The average number of infected cells, as detected by immunofluorescence in the noncultured mononuclear cell samples, was 1 to 5/300,000, and was usually > 10/300,000 in the cultured cell samples. Virus was isolated from all postinoculation blood samples obtained from 4 heifers that were seronegative at the time of inoculation, but was not isolated from any of the preinoculation samples, or from any of the postinoculation samples obtained from 2 heifers that were seropositive at the time of inoculation. When virus isolation was attempted from separated mononuclear cells in 2 heifers, 43.7% of the noncultured and 87.5% of the cultured samples had positive results.     

ULTRASONOGRAPHIC DETECTION OF FOREIGN BODIES IN SOFT TISSUES USING TURKEY MUSCLE AS A MODEL

Foreign bodies in the soft tissues are frequent problems in both veterinary and human medical practice. Radiography has been the principle tool for diagnosis, but is unable to define many foreign bodies. This study was undertaken to compare the sonographic and radiographic visualization for a variety of foreign bodies. Fresh turkey muscle (pectoral and thigh) with skin was used as the soft tissue model. Assorted foreign bodies were introduced 1 cm into the muscle. Materials examined were small wood fragments, steel wire, glass, nails, graphite, rock, BB pellets, and polystyrene plastic. Following introduction of the foreign bodies radiographs were obtained and the specimens were studied sonographically at 5.0 mHz and 7.5 mHz. All foreign bodies were visible with sonography. A bright echo with either acoustic shadowing or reverberations was present for all specimens. Radiographically metallic foreign bodies were visible but wood, some graphite, and plastic were not seen. We conclude that radiography should remain the primary imaging modality for foreign body detection because of its general availability and low cost. If radiography is negative ultrasound can detect and guide the removal of radiolucent foreign bodies.