Two immune-based methods using immortalized porcine kidney macrophages for quantifying neutralizing activity against porcine reproductive and respiratory syndrome virus-2.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes a serious infectious disease in pigs in farms worldwide. Neutralizing antibody titer is an effective index for evaluating immunity to PRRSV; however, PRRSV has different neutralizing cross-reactivity between strains. Therefore, quantitative measurement of neutralizing antibody titers against field PRRSV strains would be required to evaluate whether neutralizing antibodies in pigs could possess neutralizing activity against individual or multiple strains. Immune-based methods, such as image cytometry (ICM) and cell-based enzyme-linked immune sorbent assay (ELISA), are quantitative and can be used to evaluate many samples. Using immortalized porcine kidney macrophages (IPKMs), which are highly susceptible to infection from field PRRSV-2 strains compared with other cell lines, immune-based methods could enable the evaluation of the neutralizing activity of porcine serum against field strains of PRRSV-2 that are difficult to isolate in conventional cells. In summary, we adapted two methods, namely ICM and cell-based ELISA, to IPKMs for quantitative neutralizing antibody titer measurements. Two immune-based methods using IPKMs are adequate for quantifying neutralizing activity of porcine serum against PRRSV-2, including field strains.

A specific promoter-type in ribonuclease L gene is associated with phagocytic activity in pigs.

We have previously generated Large White pigs with high immune competence using a selection strategy based on phagocytic activity (PA), capacity of alternative complement pathway, and antibody response after vaccination against swine erysipelas. In this study, to identify the genetic changes caused by the immune selection pressure, we compared gene expression and polymorphisms in the promoter region between pigs subjected to the immune selection (immune-selected pigs) and those that were not (non-selected pigs). After lipid A stimulation, using a microarray analysis, 37 genes related to immune function and transcription factor activity showed a greater than three-fold difference in expression between macrophages derived from immune-selected and non-selected pigs. We further performed a polymorphic analysis of the promoter region of the differentially expressed genes, and elucidated the predominant promoter-types in the immune-selected and non-selected pigs, respectively, in the genes encoding ribonuclease L (RNASEL), sterile α motif and histidine-aspartate domain containing deoxynucleoside triphosphate triphosphohydrolase 1, signal transducer and activator of transcription 3, and tripartite motif containing 21. Analysis of the association between these promoter genotypes and the immune phenotypes revealed that the immune-selected promoter-type in RNASEL was associated with increased PA and was inherited recessively. Considering that RNASEL has been reported to be involved in antimicrobial immune response of mice, it may be possible to enhance the PA of macrophages and improve disease resistance in pig populations using RNASEL promoter-type as a DNA marker for selection.

Dexamethasone enhances CD163 expression in porcine IPKM immortalized macrophages.

In our previous study, we established a unique porcine macrophage cell line, immortalized porcine kidney-derived macrophages (IPKM). The purpose of the present study was to further elucidate the characteristics of IPKM. CD163 is a scavenger receptor for the hemoglobin-haptoglobin complex and is used as a phenotypic marker of anti-inflammatory M2 macrophages. The expression of CD163 is enhanced by dexamethasone (DEX), a potent steroidal anti-inflammatory drug, in human and rodent macrophages in vitro. Therefore, we investigated the effects of DEX on CD163 expression in porcine IPKM. Treatment with DEX markedly enhanced CD163 expression in the IPKM. In addition, we found that SB203580, a selective inhibitor of p38 mitogen-activated protein kinase (MAPK), blocked the effects of DEX, suggesting that the p38 MAPK signaling pathway is involved in the regulation of the DEX-induced enhancement of CD163 expression. Since CD163 is considered to be a putative receptor for the porcine reproductive and respiratory syndrome virus (PRRSV), the effects of DEX on the infection of IPKM by PRRSV were evaluated. Although the IPKM were susceptible to infection by the Fostera PRRSV vaccine strain, DEX treatment did not affect the propagation of the virus in the IPKM. This suggests that the DEX-induced enhancement of CD163 expression alone is not sufficient to facilitate the infection of IPKM by PRRSV.

Effects of Dietary Supplementation With Enterococcus faecium and Clostridium butyricum, Either Alone or in Combination, on Growth and Fecal Microbiota Composition of Post-weaning Pigs at a Commercial Farm.

Lactic acid bacteria (LAB) and butyric acid bacteria (BAB) are commonly used as probiotics in swine production. However, their combined effect on post-weaning pigs has not been assessed. Therefore, here we investigated the individual and combined efficacy of dietary Enterococcus faecium and Clostridium butyricum on the growth and gut microbiota of post-weaning pigs at a commercial farm. Four independent trials were conducted, in each of which five pens containing 10 pigs were assigned to one of five treatments: C, basal diet; L, basal diet + live E. faecium; D, basal diet + heat-killed E. faecium; M, basal diet + C. butyricum; or L+M, basal diet + live E. faecium + C. butyricum. Each trial was conducted over a 90-day period that was divided into two phases (Phase 1, days 0-40 post-weaning; and Phase 2, days 40-90 post-weaning), with the probiotics being supplemented only during Phase 1. Ten pigs in each pen were used for body weight (BW) analysis and fecal samples were collected from five or six of these pigs. In addition, the fecal samples from one randomly selected trial were used for gut microbiota analysis. We found that pigs in the L, D, and L+M treatment groups had a significantly higher BW than those in C (p < 0.05) but pigs in the L+M treatment group had a similar BW to those in the L and M groups. Furthermore, there were no significant differences in alpha diversity among the treatments but the beta diversity (weighted UniFrac distances) showed distinct clustering patterns, with pigs in C having discrete microbiota from those in all of the probiotics treatment groups except D (C vs. L, q = 0.04; C vs. M, q = 0.06; C vs. L+M, q = 0.06). These findings indicate that dietary supplementation with live or heat-killed E. faecium enhances growth performance in pigs but there is no synergistic effect when E. faecium is used in combination with C. butyricum. Furthermore, the addition of live E. faecium and C. butyricum to the diet of pigs may change the structure of the gut microbiota.

Identification of genes showing differential expression profile associated with growth rate in skeletal muscle tissue of Landrace weanling pig.

Suppression subtractive hybridization was used to identify genes showing differential expression profile associated with growth rate in skeletal muscle tissue of Landrace weanling pig. Two subtracted cDNA populations were generated from musculus longissimus muscle tissues of selected pigs with extreme expected breeding values at the age of 100 kg. Three upregulated genes (EEF1A2, TSG101 and TTN) and six downregulated genes (ATP5B, ATP5C1, COQ3, HADHA, MYH1 and MYH7) in pig with genetic propensity for higher growth rate were identified by sequence analysis of 12 differentially expressed clones selected by differential screening following the generation of the subtracted cDNA population. Real-time PCR analysis confirmed difference in expression profiles of the identified genes in musculus longissimus muscle tissues between the two Landrace weanling pig groups with divergent genetic propensity for growth rate. Further, differential expression of the identified genes except for the TTN was validated by Western blot analysis. Additionally, the eight genes other than the ATP5C1 colocalized with the same chromosomal positions as QTLs that have been previously identified for growth rate traits. Finally, the changes of expression predicted from gene function suggested association of upregulation of expression of the EEF1A2, TSG101 and TTN genes and downregulation of the ATP5B, ATP5C1, COQ3, HADHA, MYH1 and MYH7 gene expression with increased growth rate. The identified genes will provide an important insight in understanding the molecular mechanism underlying growth rate in Landrace pig breed.

Effects of oral administration of heat-killed Enterococcus faecium strain NHRD IHARA in post-weaning piglets.

Probiotic bacteria such as lactic acid bacteria (LAB) have recently received attention as candidates for alternative anti-microbial feed additives. We previously isolated Enterococcus faecium strain NHRD IHARA (FERM BP-11090, NHRD IHARA strain) and reported its probiotic efficacy. However, we have not determined the effect of oral administration of heat-killed cells of this strain. Here, we performed two experiments to investigate the effect of oral administration of the heat-killed NHRD IHARA strain on post-weaning piglets. In Experiment 1, there was a significant improvement in growth performance (P = 0.04) and increase in serum immunoglobulin A (IgA) production (P = 0.03) in the group fed heat-killed cells. These results were similar to previous results we obtained with live cells. We also found changes in serum and fecal IgA production that were unrelated to the patterns of microbiotal change. In Experiment 2, we detected a significant improvement in villus growth in the jejunum (P = 0.0002). In conclusion, oral administration of the heat-killed NHRD IHARA strain in post-weaning piglets had the same efficacy as administration of the live strain. The heat-killed NHRD IHARA strain can be used as feed additives to improve pig growth and health on commercial farms.

Multiple marker effects of single nucleotide polymorphisms in three genes, AKIRIN2, EDG1 and RPL27A, for marbling development in Japanese Black cattle.

Marbling in beef, measured by Beef Marbling Standard (BMS) number, is an economically important trait for beef cattle breeding and markets in Japan. We previously detected three single nucleotide polymorphisms (SNPs) associated with BMS number of Japanese Black in Oita prefecture: c.*188G>A in AKIRIN2, g.1471620G>T in EDG1 and g.3109537C>T in RPL27A. Here, we carried out single and multiple marker association analyses for the three SNPs in a different commercial Japanese Black population of 892 genotyped animals. The single marker analyses with the model including a single SNP showed significant associations of all SNPs with BMS number. The multiple marker analysis with the model including the main effects of the three SNPs and their interactions detected only significant main effects of g.1471620G>T and g.3109537C>T and a significant interaction between c.*188G>A and g.1471620G>T. These findings suggest the presence of inter-allelic interactions among genes affecting the development of beef marbling. For effective marker-assisted selection for BMS number, interactions among these markers need to be considered.

Characterization of probiotic properties of Enterococcus faecium NHRD IHARA isolated from porcine feces.

We examined in vitro the adhesion of Enterococcus faecium NHRD IHARA (NHRD IHARA) to porcine small intestinal mucin (PSIM) and inhibition of the adherence of enteropathogenic bacteria due to pre-incubation of PSIM with NHRD IHARA. NHRD IHARA exhibited an effective barrier function in porcine small intestinal mucus layer.

Isolation, characterization, and effect of administration in vivo, a novel probiotic strain from pig feces.

From porcine rectal swabs or feces, we isolated lactic acid bacteria and used porcine Peyer's patch cells to select them for inducibility of IgA production as an indicator of probiotic effects. The strain selected as a new probiotic was named 'Enterococcus faecium NHRD IHARA'. To verify the probiotic effects of this strain in vivo, 536 piglets at age 25 days were assigned to either the trial group, which administrated the strain, or the control group. An increase in IgA in the feces was observed at age 45 days (P < 0.05 compared with the control group); a significant increase in serum IgA was also observed at the end of the study (P < 0.01) in the trial group. In addition, significant differences between the groups in terms of body weight (P < 0.05) and average daily gain (P < 0.01) were observed. The rate of detection of swine-pathogenic Escherichia coli gene in the feces tended to be lower in the trial group than in the controls. The novel probiotic strain; E. faecium NHRD IHARA may have beneficial effects on swine growth by inducing IgA production and reducing rates of colonization by pathogens in the body.

Replicated association of the single nucleotide polymorphism in EDG1 with marbling in three general populations of Japanese Black beef cattle.

BACKGROUND: Marbling, defined by the amount and the distribution of intramuscular fat and measured as beef marbling score (BMS), is an economically important trait of beef cattle in Japan. We recently reported that a single nucleotide polymorphism (SNP), namely, c.-312A>G, in the endothelial differentiation, sphingolipid G-protein-coupled receptor, 1 (EDG1) gene was associated with the BMS level in the Japanese Black beef cattle population of Oita prefecture, with the G allele being associated with a high level of the BMS. Thus, the c.-312A>G SNP seems to be a candidate marker for marker-assisted selection. In this study, we investigated whether this association could be replicated in 3 other independent Japanese Black cattle populations and analyzed the effect of the SNP genotypes on the carcass traits other than the BMS. FINDINGS: Statistically significant differences in the BMS level were detected among the genotypes of the c.-312A>G SNP in the Japanese black beef cattle populations of Miyazaki (P = 0.0377) and Nagasaki (P = 0.0012) prefectures, and marginal difference was detected in the Kagoshima prefecture population (P = 0.0786). The G allele in the SNP was associated with an increase in the BMS level.The G allele also seemed to have a favorable influence, if any, on the carcass weight, rib eye area and rib thickness of the cattle populations. CONCLUSIONS: These findings suggest that the association of the c.-312A>G SNP with the BMS level in the Japanese Black beef cattle population was replicated in other beef cattle populations, and revealed favorable effects of the G allele on the beef productivity in the general Japanese Black beef cattle population. Thus, we concluded that the c.-312A>G SNP is useful for effective marker-assisted selection to increase the BMS level in Japanese Black beef cattle.

Association of a single nucleotide polymorphism in akirin 2 gene with marbling in Japanese Black beef cattle.

BACKGROUND: Marbling defined by the amount and distribution of intramuscular fat, so-called Shimofuri, is an economically important trait of beef cattle in Japan. The c17-25 expressed sequence tag (EST) has been previously shown to possess expression difference in musculus longissimus muscle between low-marbled and high-marbled steer groups, and to be located within genomic region of a quantitative trait locus for marbling. Thus, the akirin 2 (AKIRIN2) gene containing the c17-25 EST sequence was considered as a positional functional candidate for the gene responsible for marbling. In this study, we explored single nucleotide polymorphism (SNP) in the AKIRIN2 and analyzed association of the SNP with marbling. FINDINGS: A SNP in the 3' untranslated region of the AKIRIN2, referred to as c.*188G>A, was the only difference detected between high- and low-marbled steer groups. The SNP was associated with marbling in 3 experiments using 100 sires (P = 0.041), 753 paternal half-sib progeny steers from 4 sires heterozygous for the c.*188G>A (P = 0.005), and 730 paternal half-sib progeny steers from 3 sires homozygous for the A allele at the c.*188G>A (P = 0.047), in Japanese Black beef cattle. The effect of genotypes of the SNP on subcutaneous fat thickness was not statistically significant (P > 0.05). CONCLUSION: These findings suggest that the AKIRIN2 SNP polymorphism is associated with marbling and may be useful for effective marker-assisted selection to increase the levels of marbling in Japanese Black beef cattle.

Association of a single nucleotide polymorphism in titin gene with marbling in Japanese Black beef cattle.

BACKGROUND: Marbling defined by the amount and distribution of intramuscular fat is an economically important trait of beef cattle in Japan. We have recently reported that single nucleotide polymorphisms (SNPs) in the endothelial differentiation, sphingolipid G-protein-coupled receptor, 1 (EDG1) gene were associated with marbling in Japanese Black beef cattle. As well as EDG1, the titin (TTN) gene, involved in myofibrillogenesis, has been previously shown to possess expression difference in musculus longissimus muscle between low-marbled and high-marbled steer groups, and to be located within genomic region of a quantitative trait locus for marbling. Thus TTN was considered as a positional functional candidate for the gene responsible for marbling. In this study, we explored SNP in TTN and analyzed association of the SNP with marbling. FINDINGS: A SNP in the promoter region of TTN, referred to as g.231054C>T, was the only difference detected between high- and low-marbled steer groups. The SNP was associated with marbling in 3 experiments using 101 sires (P = 0.004), 848 paternal half-sib progeny steers from 5 sires heterozygous for the g.231054C>T (P = 0.046), and 820 paternal half-sib progeny steers from 3 sires homozygous for C allele at the g.231054C>T (P = 0.051), in Japanese Black beef cattle. The effect of genotypes of the SNP on subcutaneous fat thickness was not statistically significant (P > 0.05). CONCLUSION: These findings suggest that in addition to the EDG1 SNPs, the TTN SNP polymorphism is associated with marbling and may be useful for effective marker-assisted selection to increase the levels of marbling in Japanese Black beef cattle. Further replicate studies will be needed to confirm the allelic association observed here, and to expand the results to evaluate all possible genotypic combinations of alleles.

Novel SNP in 5' flanking region of EDG1 associated with marbling in Japanese Black beef cattle.

Marbling, defined by the amount and distribution of intramuscular fat, is an economically important trait of beef cattle in Japan. The endothelial differentiation, sphingolipid G-protein-coupled receptor, 1 (EDG1) gene has been considered as a positional functional candidate for the gene responsible for marbling. We have recently reported that 2 single nucleotide polymorphisms (SNPs), c.-312A>G in the 5' untranslated region (UTR) and c.*446G>A in the 3' UTR in EDG1 were associated with marbling in Japanese Black beef cattle, but this was not functional and a causal mutation for marbling. In the present study, we detected 2 novel SNPs, referred to as g.1475435G>A and g.1471620G>T, in the 5' flanking region of the EDG1 between low-marbled and high-marbled steer groups, which were previously shown to have EDG1 expression differences in musculus longissimus muscle. The g.1475435G>A SNP seemed not to segregate in Japanese Black beef cattle. The g.1471620G>T SNP was associated with the predicted breeding value for beef marbling standard number by the analyses using Japanese Black beef cattle population. Based on these findings, we hypothesized that the g.1471620G>T SNP might have an impact on EDG1 expression and also marbling.

Association of a single nucleotide polymorphism in ribosomal protein L27a gene with marbling in Japanese Black beef cattle.

Marbling, defined by the amount and distribution of intramuscular fat, is an economically important trait of beef cattle in Japan. The c2-11#2 expressed sequence tag (EST) has been previously shown to possess expression difference in musculus longissimus muscle between low-marbled and high-marbled steer groups, and to be located within genomic region of a quantitative trait locus for marbling. Thus, the ribosomal protein L27a (RPL27A) gene containing the c2-11#2 EST sequence was considered as a positional candidate for the gene responsible for marbling. In the present study, a single nucleotide polymorphism (SNP) in the promoter region of the RPL27A, referred to as g.3109537C>T, was detected between the 2 steer groups. The SNP was associated with the predicted breeding value for beef marbling standard number by the analyses using Japanese Black beef cattle population. The effect of genotypes of the SNP on the predicted breeding value for subcutaneous fat thickness was not statistically significant. These findings suggest that the RPL27A SNP may be useful for effective marker-assisted selection to increase the levels of marbling in Japanese Black beef cattle.

Polymorphisms for 7 expressed sequence tags showing marbling-associated expression changes.

We have recently showed that the 7 expressed sequence tags (ESTs), c2-11#2, c3-3, c20-29, c22-3, c26-18#1, c26-42, and g5-10, possess expression differences in musculus longissimus muscle between low-marbled and high-marbled steer groups. In the present study, we detected single nucleotide polymorphisms (SNPs) in the 5' flanking regions of the 7 EST sequences between the 2 steer groups. A SNP for the c2-11#2 EST exhibited significantly different allelic distribution between animals with extremely high predicted breeding value for marbling and with extremely low one. The SNP in the ribosomal protein L27a (RPL27A) gene containing the c2-11#2 EST sequence may be related to changes in gene expression and/or marbling.

Polymorphisms in promoter regions of PDHB, SORBS1, and EDG1 genes showing marbling-associated expression changes.

We have recently showed that the pyruvate dehydrogenase (lipoamide) beta (PDHB) gene involved in fatty acid oxidation, the sorbin and SH3 domain containing 1 (SORBS1) gene involved in insulin-stimulated glucose uptake, and the endothelial differentiation, sphingolipid G-protein-coupled receptor, 1 (EDG1) gene involved in blood vessel formation possess expression differences in musculus longissimus muscle between low-marbled and high-marbled steer groups. In the present study, we detected single nucleotide polymorphisms (SNPs) in the promoter regions of the 3 genes between the 2 steer groups. A SNP in the EDG1 exhibited significantly different allelic distribution between animals with extremely high predicted breeding value for marbling and with extremely low one. The EDG1 SNP may be related to changes in gene expression and/or marbling.

Genome-wide analysis of spatial and temporal gene expression in rice panicle development.

The basic structure of a rice inflorescence (the panicle) is determined by the pattern of branch formation, which is established at the early stages of panicle development. In this study we conducted global transcriptome profiling of the early stages of rice panicle development from phase transition to floral organ differentiation. To generate a meristem-specific gene-expression profile, shoot apical meristems (SAMs) and subsequently formed, very young panicles were collected manually and used for cDNA microarray analysis. We identified 357 out of 22,000 genes that are expressed differentially in the early stages of panicle development, and the 357 genes were classified into seven groups based on their temporal expression patterns. The most noticeable feature is that a fairly small number of genes, which are extensively enriched in transcription factors, are upregulated in the SAM immediately after phase transition. In situ hybridization analysis showed that each gene analysed exhibits a unique and interesting localization of mRNA. Remarkably, one of the transcription factors was proven to be a close downstream component of the pathway in which LAX, a major regulator of panicle branching, acts. These results suggest that our strategy--careful collection of meristems, global transcriptome analysis and subsequent in situ hybridization analysis--is useful not only to obtain a genome-wide view of gene expression, but also to reveal genetic networks controlling rice panicle development.