Effect of a single intrauterine administration of recombinant bovine interferon-τ on day 7 of the estrous cycle on the luteal phase length and blood profile in dairy cows.

This study tested the effect of recombinant bovine interferon-tau (rboIFN-τ) on the length of estrous cycle, luteal lifespan and side effects of rboIFN-τ in the cow. A normal estrous cycle in six non-lactating cycling Holstein cows was observed (non-treated cycle), and either 2.0 mg of liposomalized rboIFN-τ (treated cycle) or bovine serum albumin (BSA; placebo cycle) was infused in the uterus on day 7 of the estrous cycle (day 0=day of ovulation). Rectal temperature, heart rate and respiratory rate were recorded and blood samples were collected before and after the treatments. The length of the estrous cycle and corpus luteum lifespan in rboIFN-τ treated cycles were not significantly different from those of the non-treated and placebo cycles. In contrast, the rboIFN-τ treatment caused a transient increase in rectal temperature and a decrease in the number of peripheral lymphocytes and neutrophils after the treatment.

Activation of immune cells in bovine mammary gland secretions by zymosan-treated bovine serum.

Mastitis, caused by bacterial infection of the mammary gland, is a major disease of dairy cattle. The greatest risks of intramammary infection occur at the end of lactation and at the initiation of the next lactation when the cow calves. Treating serum with zymosan (yeast cell wall preparation) causes the complement to cleave, allowing this serum to serve as a source of complement fragment 5a (C5a), a potent chemoattractant and activator of the immune system. Our hypothesis was that intramammary infusion of zymosan-treated serum (ZTS) would recruit polymorphonuclear neutrophils (PMN) and generate prolonged activity in lymphocytes within the mammary gland. Ultimately this could help prevent bacterial infections in cows at dry-off and at the initiation of lactation. Two ipsilateral quarters of the mammary gland of each cow were infused with ZTS (12.5 mL/quarter), and 2 contralateral quarters were infused with saline in 8 cows shortly after lactation ended. Mammary secretions were collected periodically throughout the dry period and the first 2 wk of the next lactation. Activation status of lymphocytes and PMN in those secretions was assessed based on the intracellular presence or absence of IFN-gamma and IL-8 as determined by flow cytometry. The ZTS infusion greatly increased PMN numbers in mammary secretions for the first week only. The percentage of IFN-gamma positive lymphocytes and PMN, and the percentage of IL-8 positive PMN, exhibited a sustained increase in secretions from ZTS-treated quarters through the first 2 wk of lactation. The ZTS can stimulate PMN and lymphocyte-mediated immune defense mechanisms in the mammary gland, which may provide a useful means of preventing new intramammary infections during the dry period as well as at the initiation of lactation.

Quantitation of bovine macrophage colony-stimulating factor in bovine serum by ELISA.

We established an enzyme-linked immunosorbent assay (ELISA) system for the quantitation of bovine macrophage colony-stimulating factor (M-CSF) and used it to measure the serum M-CSF levels in bovine fetuses and calves. The average serum M-CSF level was 2.7+/-1.5 ng/ml in 39 calves under 100 days old, and 1.8+/-0.8 ng/ml in 15 cattle between 101 and 418 days old. Fetal sera samples (n = 6) prepared from cattle between 150 and 280 days of gestational age had a higher average level of M-CSF (8.8+/-1.4 ng/ml). Alteration in serum M-CSF levels in each individual calf was also measured. The serum levels of M-CSF in calves at 0-1 day after birth ranged from 0.52 to 7.3 ng/ml. During the period 113-125 days after birth, serum levels were around 1.4+/-0.39 ng/ml. Although serum M-CSF levels generally decreased as the age of calves advanced, differences among individuals, especially among newborn calves, were observed.

Mucosal immunoadjuvant activity of the low toxic recombinant Escherichia coli heat-labile enterotoxin produced by Bacillus brevis for the bacterial subunit or component vaccine in pigs and cattle.

A gene encoding the mature Escherichia coli heat-labile enterotoxin (LT) lacking the nick site in the A subunit by deleting tripeptides was introduced in a vector pNH301 and expressed extracellularly as mutant molecule of holotoxin at high levels in Bacilus brevis HPD31-S5 of the host bacterium. The mucosal adjuvant activities of the produced mutant LT (mLT) preparation were studied in pigs and cattle. Intranasal immunization of pigs with the recombinant subunit vaccine of Erysipelothrix rhusiopathiae or the component vaccine of Bordetella bronchiseptica mixed with the mLT resulted in a substantial enhancement of both mucosal and serum-specific antibody levels. The immunized pigs were also protected when challenge-exposed intradermally with a highly virulent E. rhusiopathiae strain or challenge-exposed intranasally with a highly virulent strain of B. bronchiseptica. The mLT intranasally administered with recombinant intimin (an outer membrane adhesin) of E. coli O157:H7 also induced an elevation of IgA-specific antibody in the nasal secretion and saliva of calves as well as an elevation of IgG1-specific antibody level against the intimin in the sera and colostrum of cows. The three kinds tested protein antigens were poorly immunogenic when antigen administered intranasally alone. The mLT intranasally administered at a higher effective dose did not induce local adverse reactions or diarrhea in pigs and cattle. The present study demonstrates that the recombinant mLT produced using the B. brevis expression system might represent promising immunoadjuvants for the potential application of intranasal vaccines directed against infectious diseases in pigs and cattle.

Effects of recombinant bovine interferon gamma on Strongyloides papillosus infection in calves.

The effects of interferon (IFN) gamma on the course of infection with Strongyloides papillosus in calves were investigated. Calves (N = 7 each) were inoculated with recombinant bovine IFNy or control solution daily from day 0 to day 15 following S. papillosus infection. Treatment with IFN-gamma induced an increase in faecal egg output in the peak stage of infection. The IFNgamma-treated animals harboured more worms, especially more immature worms, in the small intestine than control animals at necropsy on day 17, with no decreases in intestinal mucosal mast cells. Both animal groups had similar small numbers of intestinal worms at necropsy on day 26. All control animals developed peripheral blood eosinophilia on day 7, while five of seven IFN-gamma-treated animals did not. Serum alpha1-acid glycoprotein concentrations increased on day 7 in both animal groups, with higher values in control animals than in IFNgamma-treated animals. Control animals mounted a predominant IgG1 response to S. papillosus from day 10, while IFNgamma-treated animals did from day 22. These data suggested that IFNgamma inhibited some host protective responses to S. papillosus migrating larvae, resulting in an improvement of worm survival after a period when protective responses should be activated during the early stage of infection. The effects of IFNgamma on intestinal worm expulsion should be confirmed by further experiments.

Porcine dendritic cells generated in vitro: morphological, phenotypic and functional properties.

Despite the central role that dendritic cells (DC) play in immune regulation and antigen presentation, little is known about porcine DC. In this study, two sources of DC were employed. Bone marrow haematopoietic cell-derived DC (BM-DC) were generated using granulocyte-macrophage colony-stimulating factor (GM-CSF) in the presence or absence of tumour necrosis factor-alpha (TNF-alpha). Monocyte-derived DC (Momicron-DC) were generated with GM-CSF and interleukin-4 (IL-4). In both systems, non-adherent cells developed with dendritic morphology, expressing high levels of major histocompatibility complex (MHC) class II. The presence of TNF-alpha increased the BM-DC yield, and enhanced T-cell stimulatory capacity. Both BM-DC and Momicron-DC expressed the pan-myeloid marker SWC3, as well as CD1 and CD80/86, but were also CD14+ and CD16+. The CD16 molecule was functional, acting as a low-affinity Fc receptor. In contrast, the CD14 on DC appeared to differ functionally from monocyte CD14: attempts to block CD14, in terms of lipopolysaccharide (LPS)-induced procoagulant activity (PCA), failed. The use of TNF-alpha or LPS for DC maturation induced up-regulation of MHC class II and/or CD80/86, but also CD14. Allogeneic mixed leucocyte reactions and staphylococcal enterotoxin B antigen presentation assays demonstrated that these DC possessed potent T-cell stimulatory capacity. No T helper cell polarization was noted. Both the BM-DC and the Momicron-DC induced a strong interferon-gamma and IL-4 response. Taken together, porcine DC generated in vitro possess certain characteristics relating them to DC from other species including humans, but the continued presence of CD14 and CD16 on mature and immature porcine DC was a notable difference.

DNA mediated immunization with encoding the nucleoprotein gene of porcine transmissible gastroenteritis virus.

The immune response to a naked plasmid DNA encoding the nucleoprotein (N protein) of porcine transmissible gastroenteritis virus (TGEV) was investigated in this study. A complementary DNA of the entire N gene was amplified by RT-PCR, and inserted into a mammalian expression vector (pcDNA3.1) to construct a recombinant plasmid (pcDNA/N). To evaluate the immunogenicity of the construct, BALB/c mice were intramuscularly immunized with different doses (50, 100 and 200 microg/mouse) of pcDNA/N twice at a 5-week interval. An optimal antibody response was achieved with 100 microg of pcDNA/N. The response lasted at least 11 weeks after primary immunization. By western blotting analysis, the antibodies specifically recognized a 47 kDa protein corresponding to the viral N protein, but they did not reveal neutralizing activity against infectious TGEV in vitro. Immunoglobulin G2a was predominant among these antibodies, which was indicative of Th1 type cell activation in pcDNA/N immunized mice. Moreover, spleen cells from these mice showed stronger immune responses than those from live vaccine or parental vector immunized mice. These results suggest that the construct can elicit both humoral and cell-mediated immune (CMI) responses against TGEV N protein in mice.

Molecular cloning, characterization, and expression of porcine Fas ligand (CD95 ligand).

We isolated and sequenced cDNA that contained the coding sequence of porcine Fas ligand (FasL). Using mixed oligonucleotide primers based on the 5' and 3' nucleotide sequences conserved among human, murine, and rat FasL, we performed the reverse transcription polymerase chain reaction (RT-PCR) with total RNA prepared from porcine thymocytes stimulated with 5 microg/ml concanavalin A (ConA) to clone the cDNA of porcine FasL. The open reading frame (ORF) of porcine FasL cDNA was 849 base pairs (bp) in length and encoded 282 amino acids. The predicted amino acid sequence was 85.5%, 76.6%, and 75.5% homologous to the predicted human, murine, and rat FasL, respectively. The recombinant porcine FasL expressed by recombinant baculovirus containing the whole coding sequences of porcine FasL showed cytotoxic effect and induced apoptosis in porcine renal tubular cell line PK-15 cells sensitized by cycloheximide (CHX), which was confirmed by MTT assay, DNA fragmentation assay, and TUNEL staining, respectively. Furthermore, the mRNA expression of porcine FasL in porcine peripheral blood lymphocytes (PBL) was induced by porcine interleukin-18 (IL-18). These results indicate that porcine FasL identified in this study is biologically functional and has the ability to induce apoptosis as reported in other species.

Efficient production of biologically active porcine interleukin-18 by coexpression with porcine caspase-1 using a baculovirus expression system.

We previously reported that the precursor form of porcine interleukin-18 (IL-18) expressed by the baculovirus system was able to be secreted efficiently into the supernatant of insect cells, whereas only small amounts of mature IL-18 were secreted from insect cells. As insect cells do not normally have the IL-1beta converting enzyme (caspase-1), which is required for processing of the precursor IL-18 into the mature IL-18, we recently cloned porcine caspase-1 cDNA. In this study, we constructed a recombinant baculovirus containing the cDNA encoding porcine caspase-1 and showed that the coexpression of caspase-1 and the precursor IL-18 enabled insect cells to secrete mature IL-18 into the culture supernatant efficiently. Moreover, inhibition of caspase-1 activity by its specific inhibitor prevented the processing of precursor IL-18 into the mature form. These results indicated that the processing and secretion of precursor IL-18 into the mature form in insect cells were enhanced by the artificial introduction of caspase-1 activity for cleavage.

Production of biologically active recombinant bovine interferon-gamma by two different baculovirus gene expression systems using insect cells and silkworm larvae.

The full-length bovine interferon-gamma (bIFN-gamma) cDNA, including the secretion signal peptide coding region was recloned into baculovirus transfer vectors pAcYM1 and pBm050. These vectors were co-transfected with Autographa californica nuclear polyhedrosis virus (AcNPV) or Bombyx mori nuclear polyhedrosis virus (BmNPV) DNA into Spodoptera frugiperda cells (SF21AE) and Bombyx mori cells (BmN), respectively. The recombinant viruses, named AcBIFN-gamma and BmBIFN-gamma, were then recovered. Recombinant bIFN-gamma (rbIFN-gamma) was accumulated in the culture fluid of AcBIFN-gamma-infected Trichoplusia ni cells and BmBIFN-gamma-infected silkworm larvae. These rbIFN-gamma forms were shown to be glycosylated 20 and 22 kDa proteins as confirmed by SDS-PAGE and tunicamycin treatment. These products were sensitive to cystein proteinase. Both rbIFN-gamma proteins, showed high-level biological activities by plaque reduction assay using vesicular stomatitis virus, and MHC class II antigen induction on bovine macrophage cells.

A serodiagnostic ELISA using recombinant antigen of swine transmissible gastroenteritis virus nucleoprotein.

A serodiagnostic ELISA utilizing the recombinant nucleoprotein (rN protein) of transmissible gastroenteritis virus (TGEV) was developed, and evaluated by examining a panel of 141 virus neutralization (VN) positive and 101 negative sera. The rN protein-based ELISA (rnELISA) appeared to be highly sensitive and specific (98.6% and 98.0%, respectively) when it was compared to the VN test. The result was similar to that of an ELISA based on purified viral antigens with showing good correlation (R=0.829). No cross-reaction was detected with antisera against porcine epidemic diarrhea virus, hog cholera virus, type A rotavirus, pseudorabies virus and swine vesicular disease virus in this ELISA. The rnELISA can be an alternative for the diagnosis of TGE with a great advantage in antigen preparation.

Anti-viral effect of recombinant bovine interferon gamma on bovine leukaemia virus.

The antiviral activity of recombinant bovine interferon gamma (rbIFN-gamma) against bovine leukaemia virus (BLV) was evaluated by an in vitro assay. rbIFN-gamma was prepared using a baculovirus expression system and replication of BLV was measured by syncytium assay. Antiviral effects were observed when bovine and sheep cells were used as target cells or effector cells and treated with 0.1 unit/ml of rbIFN-gamma. Formed syncytium numbers were reduced less than 1/20 when these cells were treated with 10 units/ml of rbIFN-gamma. However, the antiviral effects on cells of heterologous species were decreased and more than 1000 units/ml of rbIFN-gamma were required to induce an anti-BLV effect on the combination of CC81 cells as target cells and Bat2Cl6 cells as effector cells, which originated from the cat and bat, respectively. When the degree of BLV production was estimated by reverse transcriptase (RT) activity, no antiviral effect of rbIFN-gamma was induced soon after the treatment, but it was evident in the cells persistently infected with BLV. These results showed that rbIFN-gamma suppresses the replication of BLV in vitro, but has effective biological activity on cells of homologous species.

Cloning and chromosomal assignment of the porcine interleukin-2 receptor alpha (IL-2Ralpha) gene.

Porcine genomic DNA encoding a 55 kDa subunit of interleukin-2 receptor (IL-2R), which is termed alpha chain (IL-2Ralpha), was cloned by repeated plaque hybridization using IL-2Ralpha cDNA as a probe. Two different lambda phage clones, one of which encoded exon 1 and the 5'-upstream flanking region of IL-2Ralpha gene and another encoded the sequence from exon 2 to exon 8, were isolated. By analysis of the 5'-upstream region of the gene, putative binding motifs for transcription factors such as GATA family proteins, Ikaros, NF-kappaB, NF-IL2Ralpha and SRF, were found as described in human, murine and bovine genes. Two additional motifs for STAT4 binding were also found in this region. Moreover, using the FISH technique, we assigned the porcine IL-2Ralpha locus to the distal end of the long arm of chromosome 10 (10q6-qter) where the vimentin gene had been assigned nearby.

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.

Production and characterization of monoclonal antibodies that recognize bovine Kit receptor.

Kit receptor is a transmembrane tyrosine kinase that is the receptor for stem cell factor (SCF). The extracellular domain of bovine Kit receptor (boKit) was produced by a baculovirus expression system. Six monoclonal antibody (MAb) clones designated as bK-1 to bK-6 were obtained upon immunization of mice with the recombinant protein. Immunoprecipitation and flow cytometric analysis indicated that all of the MAbs specifically bound to boKit expressed in COS-7 cells transfected with boKit cDNA. Four of the six MAbs neutralized the biological activity of recombinant bovine SCF, whereas the other two did not. The boKit-positive and boKit-negative cell fractions were sorted from cryopreserved bovine bone marrow cells by the use of MAb bK-1. Colony formation assays indicated that the cells which were able to grow in response to bovine SCF were enriched in the boKit-positive fraction. These MAbs would be valuable in studying possible boKit-positive cell species such as bovine hematopoietic cells, and in defining the biological role of Kit receptor in cattle.

Expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor on B-1a cell from persistent lymphocytosis (PL) cows and lymphoma cell induced by bovine leukemia virus.

The effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on B lymphocytes from persistent lymphocytosis (PL) cattle and lymphoma cells induced by bovine leukemia virus (BLV) was studied in vitro. Flow cytometric analysis showed that high levels of receptors to GM-CSF were expressed on these cell types. Proliferation of these B cells was induced in response to bovine GM-CSF. In tumor cell lines, the rate of cell proliferation was correlated with expression of GM-CSF receptors. A monoclonal antibody to GM-CSF inhibited lymphocyte proliferation and blocked the GM-CSF binding of lymphocytes. Cells expressing GM-CSF receptor were Ig positive and both CD5 and CD11 positive (B-1a cell). These results suggest that an abnormal expression of GM-CSF receptors on B lymphocytes from PL and lymphoma cells induced by BLV plays important roles in the PL and proliferation of lymphoma.

Production of biologically active, heterodimeric porcine interleukin-12 using a monocistronic baculoviral expression system.

A baculoviral expression system for the production of biologically active, heterodimeric interleukin (IL)-12 was developed by utilizing foot-and-mouth disease virus (FMDV) self-cleaving peptide, 2A. Recombinant porcine IL-12 (rpoIL-12) was produced by insect cells after infection with recombinant baculoviruses expressing the gene encoding a fusion protein of p35 and p40 subunits of IL-12 connected with 2A. By reducing and non-reducing SDS-PAGE analyses, it was demonstrated that rpoIL-12 had a heterodimeric structure which was resulted from 2A-dependent cleavage of the precursor fusion protein. In contrast, uncleaved, monomeric rpoIL-12 was produced by infection with baculoviruses expressing the gene lacking the 2A sequence. To assess the biological activities of these recombinants, we performed the proliferation assays of PHA-activated human PBMCs. The heterodimeric rpoIL-12 induced proliferation in a dose-dependent manner, whereas the uncleaved rpoIL-12 did not. Moreover, such biological activity was specifically inhibited by addition of anti-IL-12 antibodies or rpoIL-12 p40. These observations suggest that FMDV 2A can exert its self-cleaving activity even in a heterologous system, and that biologically active, heterodimeric rpoIL-12 can be generated by monocistronic expression of the p35/p40 fusion gene in combination with the 2A sequence.

Cloning and sequencing of cDNA encoding bovine macrophage colony-stimulating factor (bM-CSF) and expression of recombinant bM-CSF using baculovirus.

The cDNAs encoding bovine macrophage colony-stimulating factors alpha and beta (M-CSF alpha and M-CSF beta) were cloned and recombinant bovine M-CSF alpha (rbM-CSF beta) in its dimeric form was expressed by using a recombinant baculovirus/insect cell system. The predicted amino acid sequence of rbM-CSF alpha and rbM-CSF beta shared 83.3 and 75.9% (alpha), 75.3 and 65.9% (beta) similarity with the sequence for human and murine M-CSFs, respectively. The biological activity of rbM-CSF beta was confirmed by the colony-forming assay using mouse bone marrow cells. SDS-PAGE under a reducing condition showed that the molecular weight of rbM-CSF beta was approximately 34 kDa. On the other hand, Western blot analysis under a non-reducing condition revealed that this rbM-CSF beta was secreted in dimeric form into the cell supernatant.

Expression of biologically active recombinant porcine GM-CSF by baculovirus gene expression system.

The full length porcine granulocyte/macrophage colony stimulating factor (GM-CSF) cDNA, including secretion signal peptide coding region was recloned into baculovirus transfer vector pAcYM1. The vector was then transfected with Autographica californica nuclear polyhedrosis virus (AcNPV) DNA into SF21AE cells and the recombinant virus AcPGM was recovered. Recombinant porcine GM-CSF (rpGM-CSF) was obtained from the serum-free culture medium of Tn5 cells infected with the AcPGM virus, and was shown to be a glycosylated 21 kDa protein as confirmed by tunicamycin treatment and [3H]-glucosamine uptake. The biological activities of rpGM-CSF in AcPGM-infected cell culture supernatants were demonstrated by porcine bone marrow cell proliferation and haematopoietic cell colony formation assays. The use of rpGM-CSF enabled us to culture porcine monocytes/macrophage and dendritic-like cells, derived from either porcine bone marrow or peripheral blood, for up to 4 months.

cDNA cloning of porcine interleukin-2 receptor-alpha gene.

Porcine interleukin-2 receptor-alpha subunit (IL-2R alpha) cDNA was cloned from the cDNA library of Con A-stimulated PBMC. The coding sequence of porcine IL-2R alpha, including the signal peptide sequence, is 813 b.p. in length. The identities of the sequence when it was compared with ovine, murine, feline and human sequences were 72.2, 62.4, 69.8 and 68.9% at nucleotide level and 58.9, 44.6, 54.6 and 55.6% at amino acid level, respectively. Then, the coding sequence of porcine IL-2R alpha was subcloned into the COS expression vector, pcDNA3.1/Zeo(+), and transfected into COS-7 cells. The expressed protein was specifically reactive to the mAb, 231-3B2, which seemed to be specific for porcine IL-2R alpha. This result reciprocally confirmed that the mAb, 231-3B2, recognizes porcine IL-2R alpha on a molecular basis.