Normal Swine Research Articles

Effects of Digoxin on Diaphragmatic Contractility in the Piglet

Minute ventilation, arterial blood gases and pH, cardiac output, and transdiaphragmatic force generation were measured in eight anesthetized, tracheostomized piglets before and after administration of 40 micrograms/kg body wt of digoxin. No changes were noted in cardiac output, heart rate, tidal vol, respiratory rate, or minute ventilation. Blood pressure decreased from baseline to 60 min postinfusion. No change was noted in the force frequency curves at 30 and 60 min postinfusion. Serum digoxin levels averaged 10.4 ng/mL at 30 min and 5.6 ng/mL at 60 min. In seven control animals, no changes were noted in any ventilatory or hemodynamic parameter from baseline to 60 min. In contrast to the reported effects of digoxin on the diaphragm of adult humans and animals, digoxin did not augment diaphragmatic contractility in normal infant swine.

Heterogeneity of endothelium-dependent and independent responses among large and small porcine pulmonary arteries

To determine whether heterogeneity of endothelium-dependent or independent responsiveness exists between large and small pulmonary arteries, isolated rings (3–4 mm long) from large (5–7 mm in diameter) and small (2–3 mm in diameter) intralobar pulmonary arteries were prepared from normal swine and studied in vitro. Rings, with and without endothelium, were suspended in modified Krebs-Ringer bicarbonate solution, bubbled with 95% O 2-5% CO 2, maintained at 37 °C and studied in parallel. Contractions to phenylephrine, norepinephrine and histamine were significantly potentiated in small rings, with or without endothelium, compared to large pulmonary artery rings with or without endothelium. The α-adrenergic agonist, UK 14304, however, caused comparable contractions in rings without endothelium taken from large and small vessels. Contractions to potassium chloride in rings without endothelium were also comparable. Relaxant responses were assessed in rings incubated with indomethacin and contracted with phenylephrine or prostaglanding F 2α. Acetylcholine, bradykinin, the calcium ionophore A23187 and UK 14304 elicited endothelium-dependent relaxations in rings from both large and small vessels. Relaxations induced by acetylcholine and bradykinin but not those by A23187 were significantly shifted downward in small versus large pulmonary-artery rings. Isoproterenol also caused endothelium-augmented relaxations but in rings from small vessels only. Sodium nitroprusside and nitric oxide caused similar relaxations in rings from both orders. These results demonstrate that a heterogeneity in endothelium-dependent and independent responsiveness exists between large and small pulmonary arteries.

Determination of circulating levels of insulin-like growth factor II (IGF-II) in swine

A heterologous radioimmunoassay system was developed for the determination of circulating IGF-II concentrations in swine. The assay utilized a monoclonal antibody against human IGF-II (Amano Intl. Ez, VA) and bovine IGF-II (Monsanto Co., MO) as the cold standard and iodinated ligand. Serial dilutions of acid-ethanol extracted normal swine sera resulted in a curve which was parallel to the bovine IGF-II standard curve. Recovery of unlabeled standard added to extracted swine sera was 101%. Neither IGF-I nor insulin were capable of cross-reacting in this assay at levels up to 100-fold excess. Using this assay, serum IGF-II levels were determined to be significantly lower when subnormal growth hormone (GH) levels existed such as in hypophysectomized swine. However, in contrast to serum IGF-I concentrations, supranormal levels of porcine GH (pGH) did not elevate serum IGF-II concentrations after 13 wk of treatment in 25 kg hogs (initial body wt). In addition, serum IGF-II levels were reduced in fasted swine, despite a significant increase in circulating GH concentrations. Thus, although normal concentrations of GH are required for maintenance of physiological levels of IGF-II in swine, the mechanism for stimulation of IGF-II secretion is less GH-dependent than IGF-I.

A comparison of immunological methods for the detection of Trichinella spiralis antigen

Eight immunological methods all using the same monoclonal antibody reagent were compared for the detection of Trichinella spiralis antigen. These were based on: (1) the direct adsorption of the antigen to the immunoadsorbent (nitrocellulose membrane, polyvinyl chloride strip or microplate); (2) capture of the antigen by antibodies pre-sensitized on the immunoadsorbent; and (3) latex agglutination. The methods found suitable were: (a) capture radioimmunoassay (capture-RIA) (sensitivity: < 0.5 μg/ml antigen); (b) direct enzyme immunoassay (direct-ELISA) (<0.5 μg/ml); (c) tube latex agglutination test (2.2 μg/ml); and (d) direct immunodot assay (8.8 μg/ml). However, the performance of the direct-ELISA was greatly affected by the presence of each of three extraneous substances (bovine serum albumin (BSA), lipopolysaccharide (LPS), normal swine muscle homogenate (NSM)) added to the antigen sample. The direct immunodot assay was also affected by the presence of BSA or LPS, whereas both capture-RIA and the tube latex agglutination methods were affected by the presence of NSM only. The findings are discussed with a view of detecting T. spiralis larvae directly from pork samples by immunological means.

Visualization of monocyte recruitment into atherosclerotic arteries using fluorescent labelling

It has been shown that monocytes are present in early atherosclerotic lesions and in mechanically-injured arterial intima, but direct morphological tracing of specific leukocyte populations into such areas has been lacking. A method for FITC-labelling of leukocytes was therefore evaluated for monocyte studies. Monocyte (95% pure) populations were isolated from blood by counterflow centrifugation and labelled by incubation with free fluorescein isothiocyanate 1-hydrochloride (FITC) in Hank's balanced salt solution. FITC-labelled monocytes showed glass adherance, spreading and migration, as well as acid phosphatase positivity and phagocytosis for up to 20 days in tissue culture. For in vivo experiments, hypercholesterolemic (H) and normal (N) swine were bled repeatedly, and monocyte populations were isolated, labelled and reinjected. Labelled cells were found in blood samples. Animals were killed after 9 days, and formaldehyde-fixed and frozen samples of aortae were studied en face and/or sectioned and examined microscopically under fluorescence. FITC-labelled leukocytes could be found adherent to sites of thickened intima but not to normal areas. Labelled cells were also detected within atherosclerotic lesions. These results show the feasibility of the labelling technique and provide direct visualization of monocyte recruitment from the blood into atherosclerotic and lesion-prone areas.

Reduced 125I-hGH binding by serum of dwarf pigs but not by serum of dwarfed poodles

1. 1. Normal and growth-deficient poodle and swine strains were characterized for serum growth hormone-binding protein (GH-BP) content as well as other growth-related hormones, and the relationship between these factors and body size was examined. 2. 2. GH-BPs were found in all strains of pigs and poodles. Concentrations of GH-BPs (as expressed by specific bindings) did not vary among the poodle breeds, but did correlate with body size in pigs. 3. 3. Insulin-like growth factors (IGFs) I and II were decreased 71 and 44% respectively in miniature compared to standard size poodles. 4. 4. Only the Yucatan micro pig strain had reduced serum IGF-I concentrations compared to normal controls. 5. 5. Growth hormone concentrations however were normal to elevated in all micro and miniature pig strains. 6. 6. Serum triiodothyronine concentrations were reduced in Yucatan mini and micro pigs in spite of normal circulating levels of thyroxine. 7. 7. Body size reductions in the swine and dog strains are probably attributable to different primary defects of various growth related hormones or hormone receptors. 8. 8. Each species breed therefore could serve as a model for a different human growth-deficient condition.

Acute heat injury to the normal swine rectum

Intracavitary hyperthermia was applied to the rectum of normal pigs at 43 degrees, 44 degrees, 45 degrees, 46 degrees, 47 degrees and 48 degrees C for 30 min. A score of temperature-induced histological changes of each specimen was made 48 h after heating. The scores from each specimen and temperature were used for regression analysis. Judging from the regression lines of the scores obtained for rectum and oesophagus, we conclude that the thermosensitivity of the rectum is about 1 degree C higher than that of the oesophagus. It suggested that 43 degrees C/30 min could be a safe dose for normal rectum. The thermosensitivity of swine rectum is discussed.

Erythrocyte membrane ATPase and calcium pumping activities in porcine malignant hyperthermia.

To investigate possible abnormalities in erythrocyte membrane enzyme activities in the pharmacogenetic disorder MH, membrane ATPase activities have been examined in erythrocyte ghosts prepared from red blood cells of MHS and normal swine. While no differences were noted in Mg2+-ATPase activities, the (Na+, K+)-ATPase activity of MHS erythrocyte ghosts was less than that of normal ghosts. Ca2+-ATPase activity exhibited low- and high-affinity Ca2+-binding sites in both types of erythrocyte ghost. While the Km for Ca2+ was greater for normal than for MHS erythrocyte ghosts at the high-affinity Ca2+-binding site, the reverse was true at the low-affinity Ca2+-binding site. Irrespective of the type of calcium binding site occupied, the Vmax for normal erythrocyte ghost Ca2+-ATPase activity was greater than that for MHS ghosts. In the presence of calmodulin, there was now no difference between MHS and normal erythrocyte ghosts in either the Km for Ca2+ or the Vmax of the Ca2+-ATPase activity. To determine if the calcium pumping activity of intact MHS and normal pig erythrocytes differed, calcium efflux from the 45Ca-loaded erythrocytes was determined; this activity was significantly greater for MHS than for normal erythrocytes. Thus, the present study confirms that there are abnormalities in the membranes of MHS pig red blood cells. However, we conclude that these abnormalities are unlikely to result in an impaired ability of MHS erythrocytes to regulate their cytosolic Ca2+ concentration.

Acute calcium homeostasis in MHS swine.

To elucidate a pathogenesis for the reduction in bone calcium content observed in MHS individuals, we studied the acute calcium homeostasis of MHS swine. This was achieved by the serial measurement, with a calcium selective electrode, of calcium transients in Landrace MHS (five) and control Landrace/large white cross MH negative (five) swine following IV bolus injection of calcium gluconate 0.1 mmol X kg-1--a dose which induced an acute 45 per cent increase in plasma ionised calcium. Experimental animals were anaesthetised with ketamine 10 mg X kg-1 IM, thiopentone (intermittent divided doses) 15-25 mg X kg-1 (total) IV and N2O/O2 (FIO2 0.3) by IPPV to maintain a normal blood gas, acid/base state. The plasma ionised calcium decay curve observed in MHS swine did not differ from that of control normal swine. Further it was noted that the induced acute rise in plasma ionised calcium failed to trigger the MH syndrome in any MHS swine. It is concluded that the mechanisms of acute calcium homeostasis in MHS swine are normal. An explanation for the reduction in bone calcium content observed in MHS individuals must be sought, therefore, through study of the slow long-term component of the calcium regulatory process. In addition, the conventional strictures placed on the use, in MHS patients, of calcium gluconate are called in question.

Method for detection and isolation of cholesteryl ester-containing "foam" cells using flow cytometry.

Accumulation of cholesteryl ester within vascular cells is a defining characteristic of atherosclerotic lesions. Therefore, it is of interest to be able to monitor this critical event in the development of atherosclerosis. With this objective in mind, we have developed a method for the detection of cholesteryl ester-containing cells (i.e., foam cells) in cell suspensions prepared from enzymatically dissociated aortas. Cholesteryl ester in aortic cells was selectively stained with the fluorescent dye filipin. Because filipin binds to unesterified cholesterol but not to esterified cholesterol, it was necessary first to remove unesterified cholesterol from cells by ethanol extraction so that its presence would not interfere with the specific detection of cholesteryl ester. Then unesterified cholesterol made available by enzymatic hydrolysis of cellular cholesteryl ester could be specifically stained with filipin. The filipin-stained cell suspensions were analyzed using flow cytometry. With a flow cytometer it was possible to detect and sort cholesteryl ester-containing cells onto glass slides for microscopic analysis. Cell suspensions prepared from either grossly normal or atherosclerotic swine aortas contained cells with cholesteryl ester inclusions. As expected, these cells were more numerous in the atherosclerotic aortas. Cells with higher levels of fluorescence contained more numerous cholesteryl ester inclusions. Flow cytometric detection of cholesteryl ester-containing cells should be generally useful in studies of cellular cholesterol metabolism as well as in specific studies of cellular cholesterol accumulation in atherosclerotic vessels.

Thermodamage, thermosensitivity and thermotolerance of normal swine oesophagus.

The normal swine oesophagus was heated using an intracavity microwave radiator for 30 min at 45, 46, 47, 48 and 49 degrees C. The animals were sacrificed 48 h or 30 days later. The degree of heat injury was evaluated on an histological score system with a maximum possible damage score of 100 per cent. The acute heat injury (48 h after heating) at these temperatures was 2, 17, 40, 50 and 67 per cent respectively. The main feature of acute heat damage was degenerative necrosis or thrombosis of blood vessels at the oesophageal wall. This occurred only in one out of nine cases at 45 and 46 degrees C heating, but in seven out of nine cases at 48 and 49 degrees C. The main feature of late heat damage was fibrosis. The score of chronic damage for the above groups was 12, 23, 30, 34 and 51 per cent, respectively. When animals were preheated at 44 degrees C for 30 min and then reheated at 45-49 degrees C 4 h later, we found that preheating could provide about 2 degrees C protection. From the above data, we suggest that 45 degrees C and 30 min heating is a safe dose for normal oesophageal tissue.

Swine chromosomes: flow sorting and spot blot hybridization.

Flow cytometry analysis was applied to swine chromosomes prepared from phytohemagglutinin (PHA) stimulated peripheral blood lymphocytes. Flow karyotypes from both sexes and from t(3;7) translocation carrier females were obtained. A certain number of chromosome pairs could be assigned to various peaks. In fact, 13 peaks were observed for 18 autosomal pairs plus X and Y. Moreover, abnormalities owing to the t(3;7) translocation were readily observable. The number of base pairs for chromosomes associated with the various peaks was estimated by comparison with human flow karyotypes. The following four peaks were thus sorted: the peak assumed to represent the translocated chromosome 7 plus the normals associated with it; the corresponding peak from a normal swine; the peak assumed to contain among others the normal chromosome 7; and finally the peak corresponding to swine chromosome 1. Chromosomes of each peak were collected on Pall Biodyne membrane. Following appropriate denaturation and prehybridization, the four samples were hybridized with a human leucocyte antigen (HLA) class I 32P-labelled cDNA probe, representing most of the coding sequence of the HLA B7 gene. The results confirmed previous data from other techniques that assigned the swine MHC(SLA) to chromosome 7. Subsequently, sorted samples were hybridized with a porcine genomic Interferon alpha probe in order to confirm the mapping of this gene family on porcine chromosome 1.

Use of dantrolene plus multiple pulses to detect stress-susceptible porcine muscle.

Twitch characteristics of tibialis anterior muscles in situ were examined in stress-susceptible and normal swine. Three groups of pigs were studied: (1) purebred Pietrain stress-susceptible, (2) purebred Yorkshire normal, and (3) a crossbred (Pietrain-Yorkshire) litter containing both stress-susceptible and normal animals. Purebred and crossbred stress-susceptible pigs provided qualitatively similar results, as did purebred and crossbred normal pigs. Single stimuli produced greater than normal peak tensions and faster rates of tension development in stress-susceptible animals. Multiple stimuli (2-6 pulses at 5-ms intervals) increased peak tensions and rates of tension development, but did not augment differences between normal and stress-susceptible pigs. Intravenous administration of dantrolene reduced peak tensions and rates of tension development in all groups for single and multiple stimuli. However, the reduction was significantly less (P less than 0.01) for stress-susceptible pigs. Multiple stimuli (4-6 pulses) plus dantrolene amplified differences (P less than 0.01) in contractile properties between normal and stress-susceptible skeletal muscles, with stress-susceptible muscles obtaining larger peak tensions and faster rates of tension development. Normal and stress-susceptible pigs may, therefore, be distinguished by these procedures.

Mycoplasma hyopharyngis, a New Species from Swine

Seven arginine-utilizing mycoplasmas isolated from upper respiratory tracts of normal swine were shown to be serologically distinct from other arginine-utilizing species previously isolated from swine. The isolates required sterol for growth but did not utilize glucose or urea. The isolates were distinct from over 80 additional mycoplasmas, as determined by epiimmunofluorescence and growth inhibition tests. Based on morphological, biological, and serological properties, we propose that these strains constitute a new mycoplasma species, Mycoplasma hyopharyngis. Strain H3-6B F (ATCC 35707) has been designated as the type strain.

Bactericidal effect of normal swine sera on Treponema hyodysenteriae.

Treponema hyodysenteriae was incubated in 20% normal swine sera (NSS) at 37 degrees C for 4 h, and viability was determined by a plate dilution method. NSS was bactericidal for nonpathogenic T. innocens and avirulent T. hyodysenteriae, but not for virulent T. hyodysenteriae isolates. Heat inactivation at 56 degrees C for 30 min, treatment with EDTA or EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N-tetraacetic acid], or removal of immunoglobulin M eliminated the bactericidal activity of NSS. However, removal of the alternate complement pathway with 10 mg of bentonite per ml did not remove bactericidal activity of NSS. Incubation of virulent isolates of T. hyodysenteriae in the presence of specific antisera plus NSS resulted in bactericidal activity. These data suggest that complement and natural antibody may be involved in protecting the host from T. innocens or avirulent T. hyodysenteriae and that T. hyodysenteriae antibody plus complement are involved in protecting convalescent pigs from re-exposure to swine dysentery.

Identification and characterization of a hapten-modifiable tepc 15 cross-reactive idiotype in swine

Rabbits and swine immunized with TEPC 15 IgA, goats immunized with T15-positive IgM and swine immunized with affinity-pure swine anti-phosphorylcholine (PC) all produce antibodies which recognize a hapten-inhibitable idiotypic determinant on swine anti-PC. The similarity in reactivity and order of inhibitability with various PC analogs of the heterologous (swine anti-TEPC 15) and isologous (swine anti-swine anti-PC) reagents indicates that they recognize a related idiotype and suggest it may be the predominant idiotype expressed on swine anti-PC antibodies. The heterologous and isologous anti-idiotypic reagents generated in this study recognize swine and mouse anti-PC but not normal swine IgM, IgG or MOPC 460. Only reactions with swine anti-PC and mouse T15-positive anti-PC proteins are hapten-inhibitable. The greater inhibitory capacity of trimethylammonium and acetylcholine than PC suggests that the idiotope(s) recognized on swine anti-PC by the anti-idiotypic reagents is integral rather than peripheral to the anti-PC binding site. The nearly exclusive IgM anti-PC response of swine to Streptococcus pneumoniae R36A and PC- Brucella have so far hindered attempts to study the isotypic distribution of the idiotype.

Differential counts by electron microscopy of cell types in normal intimal cell masses in swine abdominal aortas

Hyperlipidemic diet-induced atherosclerotic lesions show a predilection for locating at the site of normal collections of intimal cells in the swine abdominal aorta. This implies that preexisting intimal cell masses (ICM) evolve into early diet-induced lesions. This study characterizes in detail the distal abdominal aortic ICM of young normal swine as a basis for a better understanding of lesion development. Seventeen male Yorkshire swine were used, from neonates to 20 weeks of age. Only three of six neonates showed multilayered ICM, the remaining three showed scattered individual cells or a single layer of cells. All older swine had multilayered ICM at a predictable ventral site corresponding to Evans blue in vivo staining. The majority of cells counted by electron microscopy were smooth muscle (89 to 94%), the remaining were macrophages (1 to 8%) and poorly differentiated cells (3 to 5%). Weanling and older swine showed high proportions of contractile smooth muscle cells. Ergastoplasm-rich smooth muscle cells comprised nearly half of the neonatal population. Stainable lipids were demonstrable in 20-week-olds, but not in younger swine. No foam cells were seen, but cells containing a few droplets totaled 2%; macrophages comprised a high proportion of these. Pyknotic dead cells were rare, being found in the neonate and 20-week-old swine. Mitoses were observed in two ergastoplasm-rich smooth muscle cells in the neonate and one each in contractile smooth muscle cells of a 14- and a 20-week-old swine. Saddlebag-shaped, atypical-appearing cells were present in all age groups, involving all three cell types, and ranging from 1 to 3%. These represented either bridged nuclei and cytoplasm or more likely artefactual distortion of relatively normal cells.

Spontaneous and diet-induced coronary atherosclerosis in normal swine and swine with von Willebrand disease.

We have observed that pigs with impaired platelet function in the form of severe von Willebrand's disease (vWd) are resistant to spontaneous and to diet-induced aortic atherosclerosis. However, it has been reported that vWd pigs are susceptible to coronary atherosclerosis produced by balloon-induced injury of coronary arteries combined with an atherogenic diet. We have evaluated the development of coronary atherosclerosis in normal control (NC) and homozygous vWd pigs in two prospective studies: 1) as a spontaneous process in five NC and vWd pigs receiving a regular diet from the age of 3 months to 4 years; and 2) in nine NC and five vWd receiving a high-fat and high-cholesterol (2%) diet from the age of 3 to 9 months. All of the coronary arteries were analyzed postmortem in 5-mm sections. None of the NC nor the vWd pigs in the spontaneous study showed coronary atherosclerosis or myocardial lesions. In the study of diet-induced atherosclerosis, only one NC and one vWd pig had discrete stenoses; the stenoses affected the three coronary arteries and were significant (50% to 80%) in the NC and mild (greater than 25%) in the vWd pigs; no pigs showed myocardial lesions. Pigs with vWd are resistant to atherosclerosis of the aorta. To assess the resistance or susceptibility to coronary disease in these pigs, a longer follow-up study would be necessary.

牛と豚の各疾患時におけるNBT test成績について

The Nitroblue tetrazoliumdye (NBT) test was carried out in cattle and swine affected with various diseases.The NBT positive rate was 4-8% in normal cattle and swine. It decreased in cattle infected with infectious bovine rhinotracheites virus. It was more than 15% in cattle infected with Haemophilus somnus or some other bacteria. It decreased in swine infected with hog cholera virus. It was more than 15% in swine infected with Pasteurella, Haemophilus or some other bacteria.

Control of monocyte recruitment by chemotactic factor(s) in lesion-prone areas of swine aorta.

Aortic areas predisposed to early atherosclerosis in swine demarcated by Evans blue uptake (blue areas) show preferential intimal penetration by blood monocytes before and during lesion formation. These cells are thought to be the major source of foam cells in these early lesions. To examine mechanisms controlling monocyte migration into these areas, extracts of aortic tissue from both blue and white areas of hypercholesterolemic (H) and normal (N) swine were tested for chemotactic activity against monocytes from N and H swine and neutrophils from H swine. The data indicate that extracts of blue areas from H swine contained at least one, and possibly two, factors chemotactic only for monocytes from H swine, but not N swine. These factors were not present in similar extracts from white areas of H swine, or either blue or white area extracts from N swine. Gel filtration chromatography of crude blue area extracts separated two chemotactically active fractions of molecular weights 68,000 and 5000 that were not present in white area extracts and that elicited a positive chemotactic effect only for H swine monocytes. These findings provide a mechanism explaining our previous findings of preferential monocyte adhesion and intimal penetration in lesion-prone areas, and indicate that control of monocyte recruitment into the artery involves not only alteration of the arterial wall, but also functional changes in the circulating monocyte.