The effect of recombinant swine interleukin-4 on swine immune cells and on pro-inflammatory cytokine productions in pigs

The in vitro effect and the in vivo influence of recombinant swine IL-4 (rSwIL-4) were characterized in various swine cells and in nursery pigs on LPS-induced endotoxic shock and pro-inflammatory cytokine productions. In in vitro experiment, the rSwIL-4 induced a proliferation of CD4 positive T cells in mitogen-prestimulated peripheral blood mononuclear cell (PBMC). In addition, the rSwIL-4, which was produced from insect cells, promoted the differentiation of monocytes into immature dendritic cells in combination with granulocyte macrophage-colony stimulating factor (GM-CSF). Furthermore, the rSwIL-4 successfully suppressed the LPS-induced secretion of TNF-alpha, IL-1alpha, IL-6, IL-8, and IL-18 from swine alveolar macrophages when rSwIL-4 was treated at the same time with LPS. In in vivo experiment in nursery pigs, subcutaneous pretreatment of rSwIL-4, which was produced from baculovirus expression system, enhanced the severity of respiratory failure with endotoxic shock, and increased the production of TNF-alpha and IL-18 in response to inoculation with LPS. These results indicate that the rSwIL-4 is biologically active in both in vitro and in vivo treatments. Depending on the administration time, pro-inflammatory cytokine productions by IL-4 can cause either inhibitory or stimulatory regulation.     

Histological and immunohistochemical evaluation of granulosa cells during different stages of folliculogenesis in bovine ovaries

Bovine granulosa cells (GC) vary in their morphological aspect during different stages of folliculogenesis. In this study, 10 morphologically normal bovine ovaries were collected to study the structural aspects of different stages of GC using intermediate filament protein antibodies including cytokeratin AE1/AE3 (AE1/AE3), vimentin, nectin‐4 and desmin. Hormonal immunolocalization was assessed using the immunomarkers anti‐Müllerian hormone (AMH) and inhibin alpha. In addition, tumour markers and proliferation markers using c‐erbB‐2 oncoprotein and proliferating cell nuclear antigen, respectively, were investigated. The immunolabelling of AE1/AE3 in GC was strongest in the early follicle stage and gradually decreased when reaching the Graafian follicle stage. Its immunolabelling increased again as the stage progressed from stage I to stage III. The immunolabelling of inhibin alpha was inversely proportional to that of AE1/AE3 in the developing ovarian follicles as their immunolabelling is opposite to each other during folliculogenesis. AMH was immunopositive in almost all GC stages in different intensities and percentages, except for some negative staining in the atretic IV follicles. The atretic IV follicle is a unique type of atretic follicle that shows Call‐Exner body formation, which was mainly found in older cows in this study. The distinct patterns of immunoreactivity for various types of immunomarkers in the different GC stages will play an important role in diagnostic assistance of various follicle conditions, including cystic ovaries and GC tumours.     

Sequence of the domestic duck (Anas platyrhynchos) growth hormone-encoding gene and genetic variation in the promoter region

The duck growth hormone encoding gene and its promoter region were amplified by polymerase chain reaction (PCR). A total of 5.25 kb were cloned and sequenced. Duck growth hormone (GH) consists of five exons and four introns and is structurally similar to mammalian and chicken GH gene. Although the distal region of duck GH promoter showed no similarity to chicken and turkey promoters, the proximal region of the promoter contained two putative Pit‐1 binding sequences, and showed similarity to chicken and turkey GH promoters. Genetic variation was detected at five positions of the promoter region. The results of this study indicate that the expression of duck GH is likely regulated in a similar manner to that of chicken GH via enhancer‐type cis‐acting elements and the presence of genetic variation in the duck GH gene may be applicable to marker‐assisted selection.     

Hog Cholera: I. Investigation of the Agar Double-Diffusion Precipitation Test for The Detection of the Virus in Swine Tissue

The agar double-diffusion precipitation test was investigated to detect hog cholera virus in splenic and pancreatic tissues of swine. Special attention was given to the conditions influencing the sensitivity and specificity of the test. These studies emphasize the strict techniques and methods required in the test in order to detect specific antigen — antibody reaction. Absorption studies performed on serum fractions prepared by DEAE cellulose chromatography and studied electrophoretically, indicated the specific reaction was given by fractions, migrating in the gamma-globulin region and was absorbed by infected but not by normal spleens. The sensitivity of the test is dependent to a great extent on the successful liberation of the viral antigen from the tissue.     

Hyaluronidase is not essential for the lethality of Erysipelothrix rhusiopathiae infection in mice

To investigate the role of hyaluronidase in the pathogenicity of Erysipelothrix rhusiopathiae, transposon Tn916 was transferred from Enterococcus faecalis CG110 to a virulent strain of E. rhusiopathiae, and hyaluronidase-deficient mutants were isolated. A virulence assay in the mice showed that of the seven hyaluronidase-deficient mutants tested, six mutants were avirulent, but that one mutant, designated AST121, was as virulent as its parental strain. Western immunoblotting with a monoclonal antibody specific to the capsule, a major virulence factor of the organism, revealed that all of the avirulent mutants had lost the capsular antigen, whereas the mutant AST121 did not. These results suggest that the lack of virulence of the six hyaluronidase-negative mutants could be due to a loss of the capsule and that hyaluronidase does not contribute to the lethality of E. rhusiopathiae infection in mice.     

Horizontal transmission and host-virulence attenuation of totivirus in violet root rot fungus Helicobasidium mompa

Monokaryotic strains of Helicobasidium mompa were used as vectors of a mycovirus between various H. mompa isolates to examine the transmissibility of one of the mycoviruses, totivirus (HmTV1–17 virus) in the hypovirulent isolate V17 of H. mompa. The isolates that acquired HmTV1–17 virus were also examined for any alteration in the virulence. Twelve dikaryotic isolates of H. mompa, belonging to 11 mycelial compatibility groups (MCGs) and being mycelially incompatible with isolate V17, were used as recipients of HmTV1–17 virus. Two monokaryotic isolates that were mycelially incompatible with isolate V17 and all of the recipients were also used as vectors of HmTV1-17 virus between isolate V17 and the recipients. When isolate V17 and recipients were directly paired on plate media, HmTV1-17 virus was transmitted from isolate V17 into 2 of the 12 recipients (i.e., 2 of the 11 MCGs). Two monokaryotic strains were paired with isolate V17, and the monokaryotic strains that acquired HmTV1-17 virus were then used as new virus donors. When the monokaryotic strains containing HmTV1-17 virus were paired with the 12 recipients, HmTV1-17 virus was transmitted into 7 of the 12 recipients from the monokaryotic strains (i.e., 7 of 11 MCGs). Based on these results, we concluded that monokaryotic strains could act as vectors to transmit HmTV1-17 virus into H. mompa isolates. When four of the H. mompa isolates that acquired HmTV1-17 virus were used to inoculate apple rootstock Malus prunifolia, the virulence of all of the isolates was attenuated from that of their parental isolates. Moreover, because the DNA fingerprints of the fungal isolates that acquired HmTV1-17 virus were the same as those of their parental isolates, the infection with HmTV1-17 virus is considered the cause of virulence attenuation of H. mompa.     

Examination of the pathogenesis of diabetic nephropathy in OLETF rats

We conducted protein loading to examine the progression and pathogenesis of diabetic nephropathy. For this experiment, male OLETF, LETO, F344 and BN rats were used. This experiment was performed on rats between 5 and 30 weeks of age. Examination parameters included body weight, food intake, oral glucose tolerance test (OGTT), urinary protein level (UP), urinary albumin level (UA), glomerular filtration rate (GFR), kidney weights, light microscopy (LM) and electron microscopy (EM). In the protein-loaded OLETF group, the UP level was markedly increased 20 weeks or more after birth. In OLETF control group, GFR were higher than those in other strains. Glomerular hypertrophy and kidney weights were markedly increased in protein-loaded groups in OLETF rats. Thirty weeks after birth, EM showed that the number of polyethyleneimine (PEI) of the glomerular basement membrane (GBM) in protein-loaded OLETF group was significantly decreased compared to that in control group. These changes in OLETF rats were more marked in the protein-loaded group than those in the control group. LM showed that the number of exudative lesions with fibrin-cap in the protein-loaded OLETF group was significantly increased than those in control group. In OLETF rats, protein loading caused deterioration of nephropathy at 30 weeks of age. Therefore, it was demonstrated that not only blood sugar control but also protein intake factors play important roles in the deterioration of nephropathy in OLETF rats.     

Effects of recombinant bovine granulocyte-macrophage colony-stimulating factor on bovine peripheral blood neutrophil functions in vitro and in vivo

Effects of recombinant bovine granulocyte-macrophage colony-stimulating factor (rboGM-CSF) on bactericidal activity of bovine peripheral blood neutrophils in vitro and in vivo were studied. In in vitro experiment, bovine blood neutrophils were cultured for 9 hr in media containing 0.005, 0.05 or 0.5 microg/ml of rboGM-CSF. Neutrophils treated with rboGM-CSF showed significantly higher luminol-dependent chemiluminescence (LDCL) than control cells. In in vivo experiment, neutrophils isolated from cows injected 5.0 microg/kg of rboGM-CSF showed significantly higher Nitrobluetetrazolium (NBT) reduction value than that from control cows 24 hr post injection. Total leukocyte counts of cows injected rboGM-CSF sharply decreased 6 hr post injection and recovered to normal level 2 days post injection. Body temperature of these cows rose 6 hr post injection and back to normal level at 24 hr post injection. It was suggested that rboGM-CSF enhanced bactericidal activity of bovine neutrophils both in vitro and in vivo.