Comparative Studies on Electrodes for Rumen Bacteria Microbial Fuel Cells.

Microbial fuel cells (MFCs) using rumen bacteria have been proposed as a power source for running devices inside cattle. In this study, we explored the key parameters of the conventional bamboo charcoal electrode in an attempt to improve the amount of electrical power generated by the microbial fuel cell. We evaluated the effects of the electrode's surface area, thickness, and rumen content on power generation and determined that only the electrode's surface area affects power generation levels. Furthermore, our observations and bacterial count on the electrode revealed that rumen bacteria concentrated on the surface of the bamboo charcoal electrode and did not penetrate the interior, explaining why only the electrode's surface area affected power generation levels. A Copper (Cu) plate and Cu paper electrodes were also used to evaluate the effect of different electrodes on measuring the rumen bacteria MFC's power potential, which had a temporarily higher maximum power point (MPP) compared to the bamboo charcoal electrode. However, the open circuit voltage and MPP decreased significantly over time due to the corrosion of the Cu electrodes. The MPP for the Cu plate electrode was 775 mW/m2 and the MPP for the Cu paper electrode was 1240 mW/m2, while the MPP for bamboo charcoal electrodes was only 18.7 mW/m2. In the future, rumen bacteria MFCs are expected to be used as the power supply of rumen sensors.

In vivo effect of a TLR5 SNP (C1205T) on Salmonella enterica serovar Typhimurium infection in weaned, specific pathogen-free Landrace piglets.

Toll-like receptor 5 is a pattern-recognition receptor for bacterial flagellin. We previously reported that a single nucleotide polymorphism (SNP) of swine TLR5, C1205T, impairs recognition of Salmonella typhimurium (ST) flagellin and ethanol-killed Salmonella Choleraesuis (SC). In the present study, weaned, specific pathogen-free (SPF) Landrace piglets with CC, CT or TT genotypes were orally infected with ST (L-3569 strain) to determine the effect of this specific SNP on ST infection in vivo. Eighteen ST-infected piglets (six each with CC, CT, or TT) exhibited fever and diarrhea for 1 week after infection. TT piglets had the longest duration of fever. TT piglets had the greatest mean diarrhea score during the experimental period, followed by CT and CC piglets. Fecal ST shedding was greater in CT and TT pigs than CC pigs from 2 days after infection. Serum haptoglobin concentration increased in ST-infected piglets and to greater extents in CT and TT pigs than CC pigs. Daily weight gain was lower in infected pigs, particularly TT piglets, than control pigs. To the best of our knowledge, this study is the first to demonstrate that impairment of TLR recognition affects pig susceptibility to disease in vivo. Thus, piglets with the T allele of swine TLR5 (C1205T) exhibit impaired resistance to ST infection. Furthermore, elimination of the T allele of this SNP from Landrace pigs would lead to enhancement of their resistance to ST infection.

Monitoring ventral tail base surface temperature for fever detection in calves.

In this study, we investigated whether monitoring the ventral tail base surface temperature (ST) using a wearable wireless sensor could be effective for fever detection in calves with experimentally induced pneumonia after inoculation with Histophilus somni strain 2336. We found a significant difference in the changes in ST values between the control and H. somni-inoculated groups after 24 h of inoculation and detected fever; however, the rectal temperature showed a significant difference between the groups after 12 h of inoculation. When a significant difference in the ST between the two groups was observed, serum haptoglobin concentration and exacerbation of clinical score increased in the H. somni-inoculated group compared with those in the control group. Pneumonia was observed in the H. somni-inoculated group at necropsy, indicating that the changes in ST may reflect fever with inflammation caused by H. somni infection. Our results demonstrated that monitoring ST using a sensor attached to the ventral tail base can detect fever in calves and may be a useful and labor-saving tool for the health management of calves.

Allele-specific primer polymerase chain reaction for a single nucleotide polymorphism (C1205T) of swine toll-like receptor 5 and comparison of the allelic frequency among several pig breeds in Japan and the Czech Republic.

In the present study, an allele-specific primer-polymerase chain reaction (ASP-PCR) for genotyping a single nucleotide polymorphism (SNP) of swine Toll-like receptor 5 (TLR5) (C1205T; P402L) that is related to the impaired recognition of Salmonella enterica serovar Choleraesuis (SC) was developed. The allele frequencies in several pig breeds in Japan and the Czech Republic were also compared. The swine TLR5 C1205T mutation was successfully determined by ASP-PCR using genomic DNA samples in Japan that had previously been genotyped by a sequencing method. Using the PCR condition determined, genomic DNA samples from blood obtained from 110 pigs from seven different breeds in the Czech Republic were genotyped by the ASP-PCR. The genotyping results from the ASP-PCR completely matched the results from the sequencing method. The allele frequency of the swine TLR5 C1205T mutation was 27.5% in the Landrace breed of the Czech Republic compared with 50.0% in Japanese Landrace. In Japan, the C1205T mutation was found only in the Landrace breed, whereas in the Czech Republic it was found in both the Landrace and Piétrain breeds. These results indicate the usefulness of ASP-PCR for detecting a specific SNP for swine TLR5 affecting ligand recognition. They also suggest the possibility of genetically improving pigs to enhance their resistance against SC infection by eliminating or selecting this specific SNP of swine TLR5.

The effects of acute exposure to deoxynivalenol on some inflammatory parameters in miniature pigs.

Seven miniature pigs were injected intravenously with deoxynivalenol (DON) at 1 mg/kg body weight; afterward, the number of leukocytes in peripheral blood, the luminol-dependent chemiluminescence of neutrophils, the serum or plasma concentration of cytokines and acute-phase proteins were evaluated to determine the effects of acute exposure to DON on inflammatory responses. White blood cell counts were transiently increased at 3, 6, and 12 hr post-injection (PI) due to the increased number of neutrophils. The luminol-dependent chemiluminescence value of neutrophils was significantly elevated at 24 hr PI, indicating the activation of the bactericidal function of neutrophils. Significant increases of interleukin (IL)-8 and tumor necrosis factor-α at 3 hr PI and IL-6 at 6 hr PI were detected in the serum. The concentration of haptoglobin and serum amyloid A was significantly increased at 24 hr PI. These results suggest that acute exposure to DON induced a temporary recruitment of neutrophils in the peripheral blood by IL-8 and subsequent activation of the bactericidal function, and a transient increase of proinflammatory cytokines and acute-phase proteins, indicating the immunomodulatory effects of DON in pigs.

Expression and characterization of bluetongue virus serotype 21 VP7 antigen: C-terminal truncated protein has significantly reduced antigenicity.

In the present study, group-specific antigen VP7 of bluetongue virus (BTV) serotype 21 isolated from cattle in Tochigi prefecture in Japan in 1994 was characterized by sequencing and expression. Gene was amplified from cDNA synthesized on viral dsRNA using reverse-transcriptase-PCR. Nucleotide sequence of this isolate showed high similarity with other published BTV VP7 sequences. Full-length and C-terminal truncated forms of VP7 were expressed in insect cells by a baculovirus gene expression system under control of the viral polyhedrin promoter. Expression of full-length recombinant VP7 was confirmed by immunoprecipitation with VP7 specific monoclonal antibody (8A3B.6, ATCC). Recombinant proteins expressed with or without 6x His-tag showed good expression levels in TN5 cells and reacted well with the monoclonal antibody in the indirect ELISA. However C-terminal truncated VP7 with His-tag failed to react with this monoclonal antibody, while poor antigenicity was evident when it was reacted with infected bovine serum. Reduced antigenicity of the latter suggested that C-terminal truncation affects 8A3B.6 epitope construction probably via inhibition of VP7 trimer structure formation.

Salivary IgA as a useful non-invasive marker for restraint stress in pigs.

In this study, we investigated the expression of immunoglobulin A (IgA) in porcine salivary gland and its relationship with restraint stress in pigs. IgA was expressed in plasma cells in pig salivary gland, as confirmed by immunohistochemical staining. IgA was also detected in pig saliva itself by ELISA, and salivary IgA levels were increased by a restraint stress. Moreover, there was a circadian rhythm of IgA over the course of a day. These results are the first evidence of IgA expression related to stress in the pig saliva and may make IgA useful as a non-invasive biological marker to evaluate acute stress condition in the pigs.

Expression of inflammatory-related factors in porcine anterior pituitary-derived cell line.

Recent studies have shown that undifferentiated stem cells act as immunomodulators. To investigate the immunomodulatory function of the progenitor cells of the anterior pituitary gland, we attempted to establish a stem/progenitor cell line from the porcine anterior pituitary gland, and to detail its inflammatory cytokine expression. A cloned cell line from the porcine anterior pituitary gland was established and was designated as the porcine anterior pituitary-derived cell line (PAPC). PAPC expressed the mRNA of Nanog and Oct-4, and showed positive immunoreactivity for beta-catenin and Hes1 in its nucleus. PAPC grew stably by repeated passage and rapidly in the EGF and bFGF containing medium. RT-PCR showed that PAPC expressed mRNA of IL-1alpha, IL-6, IL-12, IL-15, IL-18 and TLR4. PAPC expressed S100alpha and IL-18 protein, which was localized in the marginal epithelial cells of Rathke's pouch. These results suggest that PAPC is a stem/progenitor cell and may regulate anterior pituitary cell function through an immuno-endocrine pathway.

Immune response of gnotobiotic piglets against Mycoplasma hyopneumoniae.

In this study, several cytokine responses were investigated during Mycoplasma hyopneumoniae (Mhp) infection using a gnotobiotic infection model. We found that several inflammatory cytokines (IL-1beta, IL-8, IL-18, and TNF-alpha) and an anti-inflammatory cytokine IL-10 were induced from peripheral blood mononuclear cells (PBMC) of germ-free (GF) piglets stimulated with heat killed Mhp whole antigens, but no IFN-gamma and IL-4 were induced by Mhp. After the intranasal infection of Mhp, IL-1beta, IL-8, IL-18, and IFN-gamma were also detected in the broncho-alveolar lavage fluids (BALF). The antigen-specific IFN-gamma and IL-10 responses after infection of Mhp were gradually suppressed during Mhp infection as well as non-specific immune response to concanavalin A (ConA) and lipopolysacchalide (LPS) at early stage of infection. These results suggested that Mhp infection modulates the immune response of pigs by inducing several cytokines, and causes immuno-suppression of pigs in a gnotobiotic condition.

Susceptibility of germ-free pigs to challenge with protease mutants of Salmonella enterica serovar Typhimurium.

Salmonella protease mutants, clpP and especially htrA, are candidate live oral vaccines in humans. A functional and mature immune system is, however, required to cope with them in mice. Here, we test the cytokine response of highly susceptible germ-free pigs to infection with Salmonella Typhimurium clpP and htrA mutants. Cytokine levels (IL-4, IL-10, IL-18 and IFN-gamma) were measured by ELISA in plasma and washes from the terminal small bowel 24h after oral challenge. Unlike the infection with the wild type strain, no IFN-gamma response and low IL-18 intestinal levels were found in pigs infected with the protease mutants. Despite this and regardless of partially reduced ability of htrA and clpP mutants to invade and multiply in a 3D4 porcine macrophage-like cell line, both the mutants were as virulent as was the wild type LT2 strain and caused fatal septicaemia in germ-free pigs. IFN-gamma and IL-18 response therefore did not correlate with the virulence of Salmonella Typhimurium. Our results indicate that htrA and clpP attenuations should be used with caution in populations in which an increased number of immunocompromised individuals can be expected.

Porcine Toll-like receptor 1, 6, and 10 genes: complete sequencing of genomic region and expression analysis.

Toll-like receptors (TLRs) recognize various microbial components and play key roles in activating the innate immune system. Hence, their function is important in swine infectious diseases. We completely determined 173,804 bp of nucleotide sequence of a genomic region including porcine TLR6 and the newly identified porcine TLR homologues TLR1 and TLR10. The porcine genomic structure of these genes was highly conserved in comparison with the corresponding region in humans. Analysis of their expression in porcine tissues showed differences in expression patterns between porcine TLR10 and TLR1 or TLR6. Moreover, phylogenetic analysis of the cytoplasmic regions of TLR genes suggested that the signal transduction pathway of TLR10 was different from those of TLR1 and TLR6. We also developed six polymorphic microsatellite markers within this genomic region; these markers will be valuable for association studies between TLR genes and resistance or susceptibility to infectious diseases in swine.

IL-18 expression in pigs following infection with Mycoplasma hyopneumoniae.

Little is known about the detail of the immune response during infection of pigs with Mycoplasma hyopneumoniae (Mhp). To further understand this important porcine pathogen, we examined the interleukin-18 (IL- 18) response in experimentally infected piglets. We found that large amounts of IL-18 were produced in the bronchoalveolar lavage fluids (BALF) of pigs experimentally infected with Mhp. However, the concentration of interferon-gamma (IFN-gamma) in the same BALF was negatively correlated with that of IL-18. The antibody response against Mhp was found to be associated with the IL-18 concentration in the BALF. Immunohistochemical staining revealed that both IL-18 and IL-18 receptor alpha chain (IL-18Ralpha) were present in macrophages and plasma cells in the lungs of Mhp-infected pigs. Lung mononuclear cells isolated from pneumonic lesions secreted IL-18 and prostaglandin E(2) (PGE(2)) in vitro, and PGE(2) production was enhanced by stimulation with IL-18. These results indicate that IL-18 produced in the pig lung contributes to the development of innate and acquired immune responses against Mhp as a proinflammatory cytokine rather than as an IFN-gamma-inducing factor and may be involved in immunomodulation in pigs.

Immunosuppression of xenograft rejection in porcine kidney PK15 cells by porcine IL-18.

Xenotransplantation, the transplantation of cells, tissues or organs between individuals of different species, would resolve the current shortage of organs, but rejection remains the major hurdle to successful xenotransplantation. In the present study, we analyzed mixed lymphocyte reactions (MLRs) and used 51Cr release assays in order to identify the proliferation and expansion of mouse CD8+ cytotoxic T lymphocyte cells against PK15, PK15/pIL-18 or PK15/mIL-18 cells. In addition, we identified T cell populations in mouse splenocytes and lymph node cells using two-color flow cytometry. It was found that the CD8+ T cells of xenograft recipients proliferated extensively and that the survival rates of populations of PK15/mIL-18 or PK15/pIL-18 cells were higher than untransfected controls. Moreover, CD3+ T cells were increased in mice injected with PK15 cells or PK15/pIL-18 cells but PK15/pIL-18 cell numbers were lower in lymph nodes than untransfected controls. CD8+ T cells numbers were reduced in the lymph nodes of PK15/pIL-18 injected mice. These results suggest that porcine IL-18 regulates anti-pig cellular rejection in C57BL/6 mice.

Cellular localization of IL-18 and IL-18 receptor in pig anterior pituitary gland.

Pro-inflammatory cytokine interleukin 18 (IL-18) has been proposed to have a role in modulating immuno-endocrine functions. Our previous study showed that IL-18 and IL-18 receptor (IL-18R) colocalized in somatotrophs of the bovine anterior pituitary gland, and the possibility that IL-18 acts on somatotrophs as an autocrine factor. In the present study, we investigated the localization of IL-18 and IL-18R in the pig anterior pituitary gland. RT-PCR analysis showed the expression of IL-18 and IL-18R mRNAin the pig anterior pituitary gland. Immunohistochemistry of IL-18 and specific hormones revealed the presence of IL-18 in somatotrophs, mammotrophs, thyrotrophs and gonadotrophs. IL-18R was localized in somatotrophs and thyrotrophs. Furthermore, the somatotrophs immunoreactive for IL-18 did not contain IL-18R. Thus, IL-18R and IL-18 were not colocalized in an identical somatotroph. These findings suggest that the localization of IL-18 in pig somatotrophs is different from that in bovine somatotrophs, although IL-18 closely associates with somatotrophs in the anterior pituitary glands in both species.

Genomic structure of eight porcine chemokine receptors and intergene sharing of an exon between CCR1 and XCR1.

We completely sequenced a 516,013-bp portion of the porcine genome that encompassed a cluster of genes for chemokine (C-C motif) receptors (CC chemokine receptors). We identified genes for six CC chemokine receptors (CCR1, CCR2, CCR3, CCR5, CCR9, and CCRL2) and two other chemokine receptors (CXCR6 and XCR1) in this region. Clarification of the entire structure of the region and the respective genes revealed their high conservation among human, mouse, and pig. Interestingly, much of the 5'UTR of porcine XCR1 shared an identical sequence with CCR1; this sharing does not occur in humans or mice. This finding suggests a mechanism for posttranscriptional switching of tandem-located genes in mammals that depends on alternative splicing. Furthermore, our findings contribute to analyses of lymphocyte trafficking and the functions of immune cells in pigs and other artiodactyls.

Cloning, expression analyses, and chromosomal location of porcine interleukin-18 receptor alpha chain (IL-18Ralpha).

We cloned and sequenced a cDNA that contains the coding sequence of the porcine interleukin-18 receptor alpha chain (PoIL-18Ralpha). Based on the conserved nucleotide sequences between human (HuIL-18Ralpha) and murine IL-18Ralpha (MuIL-18Ralpha), we performed reverse transcription-polymerase chain reaction (RT-PCR) with total RNA prepared from porcine peripheral blood lymphocytes (PBLs) stimulated with PoIL-12 to clone the cDNA of PoIL-18Ralpha. The open reading frame (ORF) of the PoIL-18Ralpha cDNA is 1620 base pairs (bp) in length and encodes 539 amino acids. The predicted amino acid sequence showed 68.2% and 50.2% identity to the human and murine amino acid sequences, respectively. Stimulation with concanavalin A (ConA) and IL-12, but not with IL-4, was shown to upregulate the expression of IL-18Ralpha mRNA in pig PBLs by RT-PCR analysis. Flow cytometric analysis also demonstrated that IL-18Ralpha was constitutively expressed on PoPBLs, and this expression was augmented by ConA stimulation. Furthermore, the PoIL-18Ralpha gene was mapped by fluorescence in situ hybridization (FISH) to porcine chromosome 3 (3q13-q14), near the location at which the IL-1beta gene had already been mapped. The present results will be helpful for understanding PoIL-18 and interferon gamma (IFN-gamma)-mediated T helper 1 (Th1) cell development.

Expression of interleukin-18 by porcine airway and intestinal epithelium.

In this study, we investigated the expression of interleukin-18 (IL-18) in porcine airway and intestinal epithelium. We found constitutive protein expression of precursor IL-18 in primary culture of porcine airway epithelium. Immunohistochemical staining revealed that porcine IL-18 was localized in the porcine airway epithelium and that it was significantly upregulated with experimental endotoxemia induced by Escherichia coli lipopolysaccharide (LPS) inoculation. We also confirmed by immunohistochemical staining that IL-18 was expressed in porcine intestinal epithelial cells. Moreover, the concentration of IL-18 in intestinal cell lysates of 1-day-old piglets was about 3-fold and 6-fold less than that in those of 1-month-old and 6-month-old piglets, respectively. Exogenous IL-18 was able to induce interferon-gamma (IFN-gamma) in the peripheral blood of 1-day-old piglets, whereas concanavalin A (ConA) was not able to induce IFN-gamma in the same condition. These results suggest that mucosal epithelial cells are among the major sources of IL-18 in pig and that IL-18 may be useful as a therapeutic agent for the enhancement of immune responses and as a vaccine adjuvant, especially in neonatal piglets.

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.

Cloning, expression, and tissue distribution of bovine interleukin-21.

Bovine interleukin-21 (IL-21) cDNA was cloned and sequenced from bovine peripheral blood lymphocytes (PBLs) stimulated with 10 microg/ml concanavalin A (ConA), 10 microg/ml phytohemagglutinin (PHA), and 50 ng/ml phorbol 12-myristate 13-acetate (PMA) for 48 h. The open reading frame of the bovine IL-21 cDNA is 459 bp in length and encodes 152 amino acids. The predicted amino acid sequence is 78.2 and 58.5% homologous to the human and murine IL-21 amino acid sequences, respectively. Recombinant bovine IL-21 was expressed by a baculovirus expression system. The bovine IL-21 was processed to the mature form in insect cells and secreted to the supernatant confirmed by N-terminal amino acid sequencing. The recombinant bovine mature IL-21 induced the proliferation of human IL-2-dependent cells, ILT-MAT. The mRNA expression for bovine IL-21 was observed in the spleen, but not in the brain, heart, lung, liver, and kidney. The bovine IL-21 identified in this study may provide new methods for the enhancement of innate immunity in cows.

High level expression and purification of bioactive bovine interleukin-18 using a baculovirus system.

Bioactive recombinant bovine interleukin-18 (rboIL-18) was expressed using a baculovirus system. Normally, IL-18 is translated as a precursor form of a 24kDa polypeptide and processed by IL-1beta converting enzyme (ICE) to a mature bioactive form of 18kDa protein. Hence, to express active form IL-18, we constructed two recombinant baculoviruses containing boIL-18 and human ICE (hICE) genes, respectively, and superinfected these viruses into insect cells. Superinfection of both recombinant viruses into the cells resulted in the expression of a 24kDa precursor form and an 18kDa mature form detectable in the supernatant by immunoblotting using anti-porcine IL-18 antibody. Culture supernatant from the superinfected cells showed a synergistic effect with recombinant boIL-12 for production of interferon-gamma (IFN-gamma) in bovine peripheral mononuclear cells. By addition of histidine hexamer at the C-terminal of boIL-18, the mature IL-18 was purified. Bioactivity remained after purification.