Effects of intramammary infusions of interleukin-8 on milk protein composition and induction of acute-phase protein in cows during mammary involution.

The effects of interleukin-8 (IL-8) on bovine mammary functions such as milk protein secretion and the blood-milk barrier during mammary involution were evaluated. Following the final milking, recombinant bovine (rb) IL-8 (5 or 25 microg) and a saline placebo were individually infused into the left- and right-front teat cisterns of 6 cows, respectively. Three cows without treatment at the final milking were also used as controls. Mammary secretions and blood were collected at -24, 0, 10, 24, 72, 168, 336, and 720 h after infusion. In the mammary glands infused with 25 microg of rbIL-8, the increases in somatic cell counts and in the concentrations of serum albumin, IgG1 and IgG2, and the decreases in the concentrations of alpha- and beta-casein and beta-lactoglobulin were greater than in the control glands. In the mammary glands infused with 5 microg of rbIL-8, compared to the glands infused with 25 microg of rbIL-8, these changes were moderate. These results indicate that rbIL-8 impairs the integrity of the blood-milk barrier and suppresses milk-specific protein secretions. In the cows infused with 25 microg of rbIL-8, the rectal temperature and serum haptoglobin level were transiently elevated after the infusion, showing that intramammary infusion of rbIL-8 could elicit systemic inflammation.

Development of efficient method for purified recombinant bovine granulocyte-macrophage colony-stimulating factor production with baculovirus-silkworm gene expression system.

Recombinant bovine granulocyte-macrophage colony-stimulating factor (rboGM-CSF) was produced by the baculovirus-silkworm expression system. It was purified to 98% by (NH(4))(2)SO(4), followed by a three-step column chromatography with silica gel, ion exchange resin and a metal chelate column. The specific activity of purified rboGM-CSF was 1.6-6.3 x 10(6) ED(50) mg(-1). By this method, the specific activity was raised 160-fold and 21% of the expressed rboGM-CSF was recovered.

Continuous large-scale production of the cytokine CXCL8 from a novel porcine cell line.

Cytokine production from two unstimulated porcine cell lines (SL-24 and SK-L) was examined using porcine cytokine detection ELISA kits and RT-PCR. Porcine IL-1 alpha, IL-6, and CXCL8 were detected in all samples examined. In particular, the SL-24 cell line (derived from bone marrow cells of a malignant lymphoma-affected pig), produced large amounts of porcine CXCL8. Flow cytometer analysis showed the cell line to be strongly CD44 positive, and was therefore considered to be of monocyte or macrophage origin. Porcine CXCL8 production was greatest (83.86 +/- 32.33 ng/mL) at six days post-cultivation. The SK-L cell line (derived from porcine kidney) also produced CXCL8, but production was less than 1.5 ng/mL. Porcine CXCL8 from the SL-24 cell line, induced chemotactic activity in porcine neutrophils, while the production of CXCL8 from the SL-24 cell line was inhibited by dexamethasone, which suggests that the mechanism of CXCL8 production is related to an NF-kappaB binding site. The production of CXCL8 from the SL-24 cell line was enhanced by the addition of recombinant porcine IL-15, which is the first reported observation of such CXCL8 production. Cloning of the SL-24 cell line by limited dilution revealed two types of cells present in the starting population. One cell type, designated as long-form cells (LC), produced large amounts of CXCL8, while the other, designated short-form cells (SC), produced small amounts of the cytokine. The LC cells were adapted to grow in serum-free medium in which they produced large amounts of CXCL8. The large-scale production of porcine CXCL8 from the SL-24 cell line will be of value in determining the mechanism of cytokine production and as a source of naturally produced porcine CXCL8.

In vitro and in vivo effects of recombinant bovine interferon-tau on bovine leukemia virus.

The antiviral effects of recombinant bovine interferon-tau (rboIFN-tau) on bovine leukemia virus (BLV) were examined in vitro and in vivo. In the in vitro experiments, BLV titers decreased in FLK-BLV cells and in peripheral blood mononuclear cells of BLV-infected cattle treated with rboIFN-tau at a concentration higher than 10(2) U/ml. In order to examine the in vivo effects of rboIFN-tau, 10 BLV-infected cattle were subcutaneously injected with rboIFN-tau. In the first experiment, 6 cows were administrated with 10(5) U/kg body weight of rboIFN-tau 3 times per week for 4 weeks, while in the second experiment 4 cows were administrated with 10(6) U/kg body weight of rboIFN-tau 3 times per week for 3 weeks. No adverse effects were observed after the administration of rboIFN-tau. In experiment No. 1, the mean BLV titers in cattle decreased in the post-rboIFN-tau administration period compared to the pre-rboIFN-tau administration period. In experiment No. 2, the mean BLV titers in cattle decreased in the rboIFN-tau administration period. These results suggest that rboIFN-tau decreases BLV titers in vitro and in vivo and that rboIFN-tau possibly reduces the degree of BLV titer in cattle without severe side effects.

The artAB genes encode a putative ADP-ribosyltransferase toxin homologue associated with Salmonella enterica serovar Typhimurium DT104.

Many bacterial pathogens encode ADP-ribosyltransferase toxins. The authors identified an ADP-ribosyltransferase toxin homologue (ArtA, ArtB) in Salmonella enterica serovar Typhimurium (S. typhimurium) DT104. ArtA is most homologous to a putative pertussis-like toxin subunit present in Salmonella typhi (STY1890) and Salmonella paratyphi A (SPA1609), while ArtB shows homology to a hypothetical periplasmic protein of S. typhi (STY1364) and S. paratyphi A (SPA1188), and a putative pertussis-like toxin subunit in S. typhi (STY1891) and S. paratyphi A (SPA1610). The artA gene was detected from the phage particle fraction upon mitomycin C induction, and the flanking region of artAB contains a prophage-like sequence, suggesting that these putative toxin genes reside within a prophage. Southern blotting analysis revealed that artA is conserved in 12 confirmed DT104 strains and in four related strains which are not phage-typed but are classified into the same group as DT104 by both amplified-fragment length polymorphism and pulsed-field gel electrophoresis. Except for one strain, NCTC 73, all 13 S. typhimurium strains which were classified into different groups from that of DT104 lacked the artA locus. The results suggest that phage-mediated recombination has resulted in the acquisition of art genes in S. typhimurium DT104 strains.

Expression cloning of gamma interferon-inducing antigens of Mycobacterium avium subsp. paratuberculosis.

Three recombinant proteins, Map10, Map39, and Map41, produced based on nucleotide sequences obtained from the screening of Mycobacterium avium subsp. paratuberculosis genomic library expressed in Escherichia coli significantly elicited gamma interferon production in peripheral blood mononuclear cells from infected cattle. Two of these proteins were members of the PPE protein family.

Establishment of a specific radioimmunoassay for bovine interferon tau.

A radioimmunoassay (RIA) was developed for quantification of bovine interferon (bIFN) tau, conceptus secretory protein, which allows for the maintenance of the corpus luteum during early pregnancy. A cDNA coding bIFN tau was derived from cultured trophoblast cells (TBs). Recombinant (r) bIFN tau was produced in a baculovirus expression system with two different viruses. The RIA was a double-antibody competitive binding assay that used anti-bIFN tau antiserum (raised in rabbits) as the primary antibody, a radioiodinated derivative of bIFNtau as the radioactive tracer, and goat anti-rabbit IgG as the secondary antibody. The antibody did not cross-react with rbIFN alpha, recombinant human IFN beta or recombinant ovine IFN tau. The correct recovery of amounts of rbIFN tau indicated good accuracy. Serially concentrated TB conditioned media, paralleled the standard curve for bIFN tau. The intra-assay and inter-assay coefficients of variation at bIFN tau levels of 7.8 and 15.6 ng/mL were 7.1 and 8.1%, and 11.0 and 8.5%, respectively. bIFN tau was directly detected in uterine flushings obtained from cows at Day 16 of pregnancy. In summary, this assay was suitable for the measurement of bIFN tau.

Molecular cloning and expression analysis of a macrophage-colony stimulating factor receptor-like gene from rainbow trout, Oncorhynchus mykiss.

The M-CSF and its receptor (M-CSFR, CSF-1R or c-fms proto-oncogene) system were initially implicated as essential in mammals for normal monocyte development as well as for pregnancy. To allow a comparison with the M-CSF and M-CSFR system of an oviparous animal, we cloned a M-CSFR-like gene from rainbow trout (Oncorhynchus mykiss). The gene was cloned from a cDNA library of head kidney. It contained an open reading frame encoding 967 amino acids with a predicted size of 109 kDa. The putative amino acid sequence of rainbow trout M-CSFR showed 54% amino acid identity to fugu (Takifugu rubripes) M-CSFR, 52% to zebrafish (Danio rerio) M-CSFR and 40% to mouse (Mus musculus) and human (Homo sapiens) M-CSFR. The M-CSFR-like gene was constitutively expressed in head kidney, kidney, intestine, spleen and blood. The gene was detected especially in the ovary of immature female rainbow trout. These results suggest that a M-CSFR-like receptor may be involved in female reproductive tracts even in an oviparous animal like fish.

A mutant of staphylococcal enterotoxin C devoid of bacterial superantigenic activity elicits a Th2 immune response for protection against Staphylococcus aureus infection.

Staphylococcal enterotoxin C (SEC), a bacterial superantigenic exotoxin, is commonly produced by invasive Staphylococcus aureus isolates, especially methicillin-resistant strains and isolates from animal diseases. We constructed and expressed a nontoxic mutant SEC (mSEC) and investigated whether immunization with mSEC, which is devoid of superantigenic activity, can protect against S. aureus infection. Mice were immunized with mSEC and challenged with viable S. aureus. The bacterial counts in the organs of mSEC-immunized mice were significantly lower and the survival rate was higher than the corresponding values for the control group. Immunization with mSEC strongly induced the production of T-helper 2 type antibodies, immunoglobulin G1, and immunoglobulin G2b. The production of interleukin-10 (IL-10) and IL-4 was significantly greater in immunized mice challenged with S. aureus than in the control mice, whereas the production of gamma interferon (IFN-gamma) was significantly decreased in the immunized mice. The cytokine response in a spleen cell culture that was stimulated with heat-killed S. aureus or SEC showed that immunization with mSEC inhibited IFN-gamma production and up-regulated IL-10 production in vitro. Furthermore, IFN-gamma and tumor necrosis factor alpha production in vitro was significantly inhibited by sera from mSEC-immunized mice but not by sera from control mice. These results suggest that immunization with mSEC devoid of superantigenic properties provides protection against S. aureus infection and that the protection might be mediated by SEC-specific neutralizing antibodies.

Establishment of a large-scale purification procedure for purified recombinant bovine interferon-tau produced by a silkworm-baculovirus gene expression system.

We developed a procedure for the large-scale purification of bovine interferon-tau (boIFN-tau) by means of a silkworm-baculovirus gene expression system. Recombinant boIFN-tau (rboIFN-tau) was efficiently produced in the silkworm infected with boIFN-tau cDNA recombinant baculovirus and accumulated in the haemolymph. To establish a purification method suitable for mass production, we tried three crude purification methods, namely, an acidification and neutralization treatment (ANT), silica gel column chromatography (SGCC), and Blue sepharose column chromatography (BSCC) with a combination of Q-sepharose (QSC) and chelating sepharose column chromatographies (CSCC). As a result, the acidification and neutralization treatment was found to be the most efficient and cost effective. With this combination, we obtained 91% pure products. To confirm the applicability of the procedure for mass production, we inoculated 100 silkworms with the recombinant virus, and recovered about 4.55 mg (1.26 x 10(8)U/mg) of 91% pure rboIFN-tau by means of a combination of the ANT, followed by QSC and CSCC.

Sensitive and specific detection of Yersinia pseudotuberculosis by loop-mediated isothermal amplification.

We developed a loop-mediated isothermal amplification method able to detect Yersinia pseudotuberculosis strains in 30 min by using six primers designed by targeting the inv gene. This method is more sensitive than PCR and might be a useful tool for detecting and identifying Y. pseudotuberculosis.

Recombinant Ascaris 16-Kilodalton protein-induced protection against Ascaris suum larval migration after intranasal vaccination in pigs.

We recently cloned a protective antigen that is commonly expressed in Ascaris species that infect humans and pigs. We evaluated the vaccinal effects of this 16-kilodalton protein (As16) in pigs, the natural host of Ascaris suum, by intranasal immunization. Pigs that received Escherichia coli-expressed recombinant As16 (rAs16) coupled with cholera toxin (CT) had significantly elevated levels of rAs16-specific serum immunoglobulin G (IgG) and mucosal-associated IgA antibodies. rAs16 evoked a type II immune response characterized by elevated levels of interleukin-4 and -10 in the culture supernatants of peripheral blood mononuclear cells of the vaccinated pigs. An increased level of rAs16-specific serum IgG1 was also detected. Pigs vaccinated with rAs16-CT were protected from migration of A. suum larvae through the lungs, as indicated by a 58% reduction in the recovery of lung-stage third-stage larvae (L3), compared with that in nonvaccinated controls. Purified immunoglobulin from rAs16-CT-vaccinated pigs inhibited survival of infective L3 and interrupted the molting of lung-stage L3. Immunofluorescence studies revealed that this immunoglobulin bound to the digestive tracts of L3, suggesting that it might inactivate functions of the gut tissues of Ascaris species. We conclude that rAs16 is a promising mucosal vaccine candidate for pig and human ascariasis.

Generation of multinucleated giant cells in vitro from bovine monocytes and macrophages.

The generation of multinucleated giant cells (MGC) from cells of the bovine monocyte-macrophage lineage was investigated. Freshly isolated monocytes were incubated with the conditioned medium (CM) of peripheral blood mononuclear cell cultures treated with Concanavalin A for 1-4 days (CM1 to CM4). Only CM1 generated MGC despite similar concentrations of IFNgamma in all CMs. Nevertheless, MGC formation from monocytes was enhanced by adding either macrophage colony-stimulating factor (M-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF), MGC formations from macrophages were observed only when macrophages were cultured with GM-CSF plus CM. These results indicate that several mechanisms to generate MGC from bovine monocytes-macrophage lineage cells exist, and that GM-CSF is a major mediator of MGC formation in cattle.

No effect of bovine interferon-tau for control of calf diarrhea and immunomodulation in calves.

Newborn calves received a low dose of bovine interferon-tau (boIFN-tau) orally for 4 weeks and calves that had developed diarrhea received a low dose of boIFN-tau orally for 5 days. No effects of boIFN-tau were seen in the duration of the diarrhea, or in daily weight gain. Calves received a high dose of boIFN-tau subcutaneously 3 times and they were then stimulated with bovine herpesvirus type 1 vaccine. No adverse effects were observed after the administration of boIFN-tau and lymphocyte subsets from calves did not change after the stimulation. Our results suggest that boIFN-tau does not seem protecting for preventing calves from diarrhea, recovering the health of calves with diarrhea or immunomodulation, although the treatment itself is not toxic.

Effect of intramammary injection of rboGM-CSF on milk levels of chemiluminescence activity, somatic cell count, and Staphylococcus aureus count in Holstein cows with S. aureus subclinical mastitis.

The effect of intramammary injection of recombinant bovine granulocyte-macrophage colony-stimulating factor (rboGM-CSF, 400 microg/10 mL) on quarter milk levels of chemiluminescence (CL) activity, and somatic cell count (SCC) and shedding pattern of Staphylococcus aureus was investigated. Ten Holstein cows, naturally infected with S. aureus were used, with either early-stage or late-stage subclinical mastitis. Injection of rboGM-CSF caused a remarkable increase in milk CL activity with a peak at 6 h after the cytokine injection in the early- and late-stage groups. In the early-stage group, milk SCC stayed around preinjection level at 6 h, rose significantly on days 1 and 2, and was followed by a smooth and significant decline to an under preinjection level (below 200 000 cells/mL) on day 7 postinjection. Alternatively, in the late-stage group, milk SCC rose significantly at 6 h after the cytokine injection and maintained high levels thereafter. The milk S. aureus count decreased drastically by the cytokine injection in the early-stage group. The bacterial count was moderately decreased in the late-stage group, but increased back to preinoculation levels on day 7 after the cytokine injection. The results suggest that the rboGM-CSF has a potential as a therapeutic agent for S. aureus infection causing subclinical mastitis of dairy cows, if the cytokine is applied at the initial stage of infection.

Method for purifying porcine mature interleukin-18 from silkworm haemolymph.

Recombinant porcine mature interleukin-18 (rPomIL-18) has been purified from the haemolymph of silkworms. After co-infection of two recombinant baculoviruses (BmAcpVL1392-IL-18-His and BmAcpVL1392-casp-1) into the silkworm, rPomIL-18 was produced and secreted into the haemolymph. Polyethylene glycol (PEG) 6000 was added to the haemolymph at 8% (v/w) to precipitate storage proteins which would otherwise bind non-specifically to the metal chelating column and the supernatant then was applied to Sepharose bonded with Ni2+. rPomIL-18 was eluted from the column using 100 mM imidazole buffer at pH 8 with a purity of 93.6%. Approximately 5.3 mg purified rPomIL-18 was obtained from 22 ml haemolymph. It could induce interferon-gamma formation from porcine peripheral blood mononuclear cells.

The proteome expression patterns in adult Ascaris suum under exposure to aerobic/anaerobic environments analyzed by two-dimensional electrophoresis.

We investigated the protein expression patterns (the proteome) in adult Ascaris suum under exposure to aerobic and anaerobic environments in vitro using two-dimensional polyacrylamide gel electrophoresis. Adult A. suum were cultured in RPMI-1640 in the absence of oxygen (anaerobic) or in its presence (aerobic), and whole worm extracts and their excretory-secretory (ES) products were prepared for protein analysis. More than 200 protein spots resolved in two-dimensional gels. A comparison of aerobic versus anaerobic cultures revealed two aerobic and three anaerobic specific spots differentially expressed in adult worm extracts. ES products released during aerobic/anaerobic cultures also exhibited one aerobic and seven anaerobic specific spots in two-dimensional maps. One of the anaerobic specific protein spot was analyzed for its internal amino acid sequence (1-LLAFELAPHGIR-12; 1-VNTVNPGAVD-10) and the protein was predicted to be a putative dehydrogenase. These findings suggest that the availability of oxygen or its absence in the environment may influence protein expression patterns in parasitic nematodes.

Molecular characterization of a prophage of Salmonella enterica serotype Typhimurium DT104.

Isolates of the Salmonella enterica serotype Typhimurium definitive phage type (DT104) were found to contain the same prophage (designated phage ST104). The complete sequence of the DNA genome of prophage ST104 was determined. The entire DNA sequence consisted of 41,391 bp, including 64 open reading frames, and exhibited high similarity to P22 and to phage type conversion phage ST64T.

Temperature and metal ions-dependent activity of the family I inorganic pyrophosphatase from the swine roundworm Ascaris suum.

Temperature dependence, heat stability and metal ions-dependent activity were examined on the Family I inorganic pyrophosphatase (PPase) recently identified from Ascaris suum. Recombinant A. suum PPase (rAsPPase) showed an optimal activity at 55 degrees C. The rAsPPase was heat stable at 40 degrees C in the absence of added Mg(2+) and at 50 degrees C in its presence. The enzyme required divalent metal ions for its activity. The preferences for the metal ions (5 mM concentration) were in the order: Mg(2+)> Co(2+)> Cu(2+)> Fe(2+)> Zn(2+)> Mn(2+). On the contrary, enzyme activity was inhibited by Ca(2+). These findings suggest that catalytic features of AsPPase are consistent with the Family I PPases reported from a wide range of organisms.

Porcine TLR2 and TLR6: identification and their involvement in Mycoplasma hyopneumoniae infection.

We successfully cloned and sequenced porcine toll-like receptor (TLR2) and TLR6 cDNA from porcine alveolar macrophages stimulated with 10 microg/ml lipopolysaccharide (LPS). The open reading frames (ORFs) of the porcine TLR2 and TLR6 cDNA were shown to be 2358 and 2391 bp in length and to encode 785 and 796 amino acids, respectively. The predicted amino acid sequence of porcine TLR2 was 72.3% homologous to human TLR2 and 61.0% homologous to murine TLR2. That of porcine TLR6 was 74.4% homologous to human TLR6 and 66.1% homologous to murine TLR6. Porcine TLR2 and TLR6 genes were both mapped to porcine chromosome 8 (TLR2: SSC8q21.1 --> 21.5; TLR6: SSC8p11.1 --> p21.1) by fluorescence in situ hybridization (FISH) and radiation hybrid mapping. Western blot analysis confirmed that TLR2 and TLR6 proteins were both expressed in porcine alveolar macrophages. Further, antiporcine TLR2 and TLR6 antibodies synergistically blocked tumor necrosis factor-alpha (TNF-alpha) production by porcine alveolar macrophages stimulated with Mycoplasma hyopneumoniae. These results indicated that both TLR2 and TLR6 are important in the recognition of M. hyopneumoniae in porcine alveolar macrophages and will be useful in understanding innate immunity against M. hyopneumoniae.