Swine Testis Research Articles

Transmissible gastroenteritis (TGE) of swine: In vitro virus attachment and effects of polyanions and polycations

Four transmissible gastroenteritis virus (TGEV) strains (Purdue-115, D-52, 188-SG and Gep-II) and two cell lines (swine testis-ST and pig kidney-RPD) were used to study virus attachment and cell susceptibility. Virus attachment was partially thermodependent and the rate varied, depending on the strain. Identical TGEV inocula produced a higher plaque number by plaque assay in the swine testis cell line (ST) than in the pig kidney cell line (RPD) but [ 3H]uridine-labèlled virus was found associated equally well with both cell lines. A field TGEV strain (Gep-II), which was unable to multiply in cell cultures, appeared able to inhibit the attachment of radiolabelled cell-passaged virus. Therefore, the susceptibility to TGEV infection was apparently not determined at the virus-to-cell attachment stage. The attachment sites on the cell surface were specific, however, differences in TGEV attachment determinant between strains were not observed. Attachment of all the virus strains tested was enhanced by DEAE-dextran and inhibited by dextran sulfate, poly- L-lysine (PLL), poly- L-α-ornithine (PLO) and protamine sulfate.

Effect of deoxycholate, amphotericin B and fongizone on transmissible gastroenteritis coronavirus

At a concentration of 2 μg/ml, neither amphotericin B nor deoxycholate had an inactivating effect upon transmissible gastroenteritis Coronavirus in-fectivity. However, amphothericin B stimulated plaque formatin in agarose and facilitated the entry of viral RNA into swine testis cells. The combination of amphotericin B + deoxycholate inactivated virus infectivity and induced a decrease in plaque diameter. Finally, in the presence of these agents, the production of infectious virus and interferon was unchanged.

Inhibition of porcine parvovirus replication by empty virus particles.

The influence of empty porcine parvovirus (PPV) particles on viral replication was examined in cell cultures and in swine. Following extensive purification, homogeneous preparations of full and empty PPV preparations were obtained and used for in vitro and in vivo analyses. In the first in vitro experiment, swine testes cells were infected with mixtures of various ratios of empty and full (E/F) particles. The production of both intracellular and extracellular virus was markedly inhibited in the presence of empty particles. This inhibition was dependent upon the concentration of empty particles present in the mixture. In the second in vitro study, various concentrations of empty particles were added prior to full virus infection. Again, marked inhibition of progeny virus production was evident and related to the concentrations of empty particles added. Based on the results of in vitro studies, the influence of empty particles on PPV infection in swine was tested by infecting mid-term and late-term gestation swine fetuses with various E/F particle ratios. Both mid-term and late-term fetuses exposed to 0:1, 1:1 and 5:1 E/F ratios displayed gross pathological evidence of PPV infection whereas fetuses exposed to E/F ratios of 30:1 or greater were grossly normal in appearance. However, fetuses infected with 30:1, 50:1 and 300:1 E/F ratios showed evidence of virus in their tissues by DNA hybridization. Regardless of the E/F ratios, late-term infected fetuses responded with high antibody titers ranging from 1024 to 4096. The results from these studies suggested that empty particles interfered with viral replication in both cell culture and in animals.

Neutralizing secretory IgA and IgG do not inhibit attachment of transmissible gastroenteritis virus.

Secretory IgA (sIgA) and IgG from porcine milk and serum, respectively, [3H]uridine-labelled virus, swine testis and pig kidney cell lines were used to examine the neutralized virus-cell interaction. Transmissible gastroenteritis virus (TGEV), 99.99% neutralized by immunoglobulin, was able to attach to the cells. Moreover, sIgA enhanced virus attachment. However, the neutralized virus was unable to enter cells, as demonstrated by the action of proteinase K which removed it from the cell surface. It was also found that pre-attached virus was still neutralizable and that IgG and sIgA had similar TGEV-neutralizing capacities.

KBSH parvovirus: comparison with porcine parvovirus

We compared the molecular, antigenic, and pathogenic properties of KBSH parvovirus to those of porcine parvovirus (PPV) isolate NADL-8. KBSH, propagated in swine testes cells in culture, possessed two major capsid polypeptides of 83 and 64 kilodaltons that were similar in size to those of PPV. KBSH-infected cells also contained an 86-kilodalton nonstructural polypeptide that was identical in size to the PPV nonstructural polypeptide (NS-1). The KBSH polypeptides were structurally similar but not identical to the corresponding PPV polypeptides, as revealed by partial proteolysis mapping. Viral replicative-form DNA from KBSH-infected cells was similar in size to PPV replicative-form DNA and exhibited similar but not identical restriction endonuclease cleavage patterns to that of PPV replicative-form DNA. Antigenically, the two viruses were also very closely related. By using heterologous and homologous antisera, the two viruses were indistinguishable in hemagglutination inhibition and immunoprecipitation assays. However, pathogenically these viruses were dramatically different. NADL-8 caused fetal death when injected into swine fetuses in utero and viremia and high persisting antibody titers when administered orally to weaning-age swine. KBSH-inoculated fetuses were normal in appearance, and pigs orally exposed to KBSH failed to establish viremia and demonstrated only transient antibody titers. Thus, KBSH appears to be a PPV that is very closely related to a highly pathogenic PPV isolate, yet is itself nonpathogenic in swine. This reduced pathogenic potential of KBSH may be attributable to its poor ability to replicate in swine.

Purification of NADPH-cytochrome c reductate from swine testis microsomes by chromatofocusing and characterization of the purified reductase

A purified NADPH-cytochrome c reductase (NADPH: ferricytochrome oxidoreductase, EC 1.6.2.4) was prepared from swine testis microsomes by detergent solubilization followed by a procedure including chromatofocusing. The reductase was eluted at an isoelectric point of 4.8 from the chromatofocusing column. 730-fold purification was achieved with an overall yield of 1.2%. The preparation was found to be homogeneous upon polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate (SDS). Upon SDS-polyacrylamide gel electrophoresis, however, the purified preparation resolved into one major band ( M r 78 000) and two minor bands ( M r 60 000 and 15 000). The enzyme contained about 1 mol each of FMN and FAD, which were both extractable with trichloroacetic acid and also boiling water. The oxidized form of the enzyme showed the absorption spectrum of a typical flavoprotein. Aerobic reduction with NADPH resulted in conversion of the spectrum into one of the air-stable semiquinone form. The activity of the purified preparation was 26 μmol cytochrome c reduced/min per mg protein under the standard assay conditions at 22°C. The enzyme catalyzed the reaction through a ping-pong mechanism.

Crystalloids of actin-like filaments in the Sertoli cell of the swine testis.

Normal swine testes, congenital cryptorchid swine testes, and normal human tests were exposed to HMM (heavy meromyosin) after either glycerination or saponin treatment in order to determine whether the fine filaments composing the crystalloids in the Sertoli cells of the cryptorchid swine testes bind HMM to form arrowhead complexes. Short bundles of microfilaments observed in the basal part of the Sertoli cells in both normal and cryptorchid testes also bind HMM. Similar bundles of HMM-bound filaments are observed in the vicinity of spermatocytes. The periodicity of the arrowhead complexes is about 35 nm, and all arrowheads on a given filament point in the same direction. In addition, the polarity of the HMM-bound filaments in a given crystalloid or bundle is uni-directional. A mechanism for the formation of the swine crystalloids has been strongly support this hypothesis. Fine filaments of Charcot-Boettcher's crystalloid in human Sertoli cells did not bind HMM. Therefore the fine filaments of the human crystalloid are not actin-like in nature.

The Effect of Partially Hydrolyzed Casein on the Growth of Human Skin Diploid Cells in Vitro

SummaryThe effect of various partially hydrolyzed caseins (PHC) on the growth of human skin diploid (prepuce) cells cultivated in vitro has been investigated. The results indicate that PHC I (amino N/total N ratio of 0.09) contains growth-stimulating activity for prepuce cells in the absence and presence of serum. This activity of PHC I was abolished when casein was further hydrolyzed with trypsin (amino N/total N ratio of 0.36 or 0.70). It is suggested that the growth-stimulating activity of casein is attributed to the casein itself or the presence of optimal amounts of amino acids and/or polypeptide fractions. Human kidney (SWINK), swine testis (ST-92) and rabbit epidermal cells (SflEp) showed little or no growth stimulation in any of the concentrations of PHC I tested. A cell culture system to assay PHC I activity is now available which could eventually short-circuit the routine use of animal assays to test burn or other wound healing cell activity. PHC I appears to substitute for serum in growth of pr...

Crystalloids of actin-like filaments in the swine sertoli cell

Crystalloids have been observed by light microscopy in Sertoli cells of both experimentally induced and spontaneous azoospermic testes of the swine and in Sertoli cells of normal human testes. However crystalloids have not been found in normal swine testes with the light microscope. Swine crystalloids are distinguished from human crystalloids in that the diameter of the filaments is 5 nm instead of 10-15 nm. This paper confirms this distinction by showing that the filaments of swine crystalloids bind heavy meromyosin (HMM) but those of human crystalloids do not.Human testes were removed from four patients with prostate cancer, whereas normal and cryptorchid swine testes were acquired from a slaughterhouse. HMM extracted from rabbit skeletal muscle was dissolved in 10 mM Tris buffer (pH 7. 4) containing 0. 1 M KCI and 0. 001 M MgCl2 at a final concentration of 7 mg/ml. Pieces of testicular tissue were exposed to HMM at 4°C for 24 hours after glycerination or saponin treatment.

Actin-like filaments in the myoid cell of the testis.

Microfilaments in the myoid cells of the peritubular tissue in the mouse, swine and human testis bind heavy meromyosin (HMM) and form arrowhead complexes. The periodicity of the arrowhead complexes is about 35 nm. Individual filaments show arrowheads that point in the same direction. Opposing polarity of the HMM-bound filaments is also observed. The microfilaments do not bind HMM in the presence of 10 mM ATP. After treatment with the contraction medium of Hoffmann-Berling, the filaments appear to be undulated. These observations indicate that the microfilaments in the myoid cell are actin-like in nature. A small number of thicker filaments (about 10 nm in diameter) which do not bind HMM is also observed in the cell. Microfibrils which have been reported around the human myoid cell are also found in the swine.

Actin-like filaments in the Sertoli cell junctional specializations in the swine and mouse testis.

Microfilaments at the junctional specializations between adjacent Sertoli cells and between the Sertoli cell and the late spermatid of the mouse and swine testes bind HMM and form arrowhead complexes with a periodicity of about 35 nm. The arrowhead formation is inhibited when the tissues are treated with HMM in the presence of ATP. These observations show that the microfilaments are actin-like in nature. The functional significance of these filaments in the Sertoli cell is discussed.

Partial characterization of the principal soluble antigens associated with the coronavirus of transmissible gastroenteritis by complement fixation and immunodiffusion

A microtiter complement fixation (CF) test to detect transmissible gastroenteritis (TGE) viral antigen was developed, using TGE hyperimmune pig serum as an antibody source. Sera from TGE covalescent pigs did not fix complement by this test. Maximal virus and soluble antigen (SA) titers were obtained 36 to 48 h after inoculation of swine testes cells. Cell-associated virus and SA titers were higher than those in the culture fluid, which had to be concentrated 20X before use as antigen in agar immunodiffusion tests (ID). By sucrose density-gradient centrifugation, the SA had a buoyant density of 1.10 g/ml and could be separated from the virus that banded in the 1.19-g/ml region. Virus and SA from three different isolates of TGE had the same buoyant densities. Heating and proteolytic enzyme digestion established the protein nature of the SA. As assayed by CF and ID, there were stability differences between crude and purified preparations of SA. Antibody prepared in rabbits against the SA neutralized the TGE virus.

Prostaglandins in swine testes.

AbstractThe prostaglandin content of swine testes was investigated. It was found that PGE1, PGE2, PGF1 and PGF2 were present, and the amount of total PGE's and PGF's was determined.