Community structure of denitrifying and total bacteria during nitrogen accumulation in an ammonia-loaded biofilter.

AIMS: To obtain insight into the complex behaviour of denitrifying and total bacterial groups during the nitrogen accumulation process in an ammonia-loaded biofiltration system. METHODS AND RESULTS: Denitrifying and total bacterial communities in a laboratory-scale rockwool biofilter with intermittent water recirculation were analysed by using denaturing gradient gel electrophoresis targeting nosZ and metabarcoding sequencing of the 16S rRNA gene. Gene abundance was evaluated by quantitative PCR. The nosZ number increased from 6·59 × 106 to 3·33 × 108 copies per gram dry sample over the 436 days of operation, during which nitrogen mass balance errors increased to 39%. The nosZ sequences associated with the genera Castellaniella, Hyphomicrobium and Pseudomonas were detected. Metabarcoding sequencing analysis indicated that the proportions of the genera for which at least one denitrifying strain or species possessing nosZ had been characterized corresponded well to the nitrogen loss. In addition, the genus Nitrosococcus (γ-proteobacteria) increased its relative abundance at days 317 and 436. CONCLUSIONS: The increased proportion of denitrifying bacteria in this ammonia-loaded biofiltration system could be related to the nitrogen loss. SIGNIFICANCE AND IMPACT OF THE STUDY: These results will help to clarify the complex behaviour of nitrifiers and denitrifiers within ammonia-loaded biofiltration systems.

Wild boars from Sweden, Austria, the Czech Republic and Japan possess intact mannose-binding lectin 2 (MBL2) genes.

The two-nucleotide deletion recently detected in the mannose-binding lectin 2 gene in purebred and crossbred domestic pigs was not found among 68 wild boars representing 4 populations from Europe and Asia. This suggests that the deletion is a result of breeding and/or genetic drift/bottle necks.

Genome-wide association QTL mapping for teat number in a purebred population of Duroc pigs.

Because of increasing litter size in Western pig breeds, additional teats are desirable to increase the capacity for nursing offspring. We applied genome-wide SNP markers to detect QTL regions that affect teat number in a Duroc population. We phenotyped 1024 animals for total teat number. A total of 36 588 SNPs on autosomes were used in the analysis. The estimated heritability for teat number was 0.34 ± 0.05 on the basis of a genomic relationship matrix constructed from all SNP markers. Using a BayesC method, we identified a total of 18 QTL regions that affected teat number in Duroc pigs; 9 of the 18 regions were newly detected.

Lactobacillus helveticus SBT2171, a cheese starter, regulates proliferation and cytokine production of immune cells.

Consumption of a Lactobacillus helveticus SBT2171 (LH2171)-containing cheese has been reported to exhibit immunoregulatory actions, including an increase in regulatory T cell population and reduction in proinflammatory cytokine production in mice. We examined the in vitro effects of LH2171 cells per se on immune cell function, specifically proliferation and cytokine production, which are primary reactions of the immune response. Immune cell fractions were prepared by mechanical disruption of mesenteric lymph nodes (MLN), Peyer's patches (PP), and spleens (SP) of mice. The cell fractions were dispensed into a culture plate and stimulated with anti-CD3/CD28 antibody beads in place of antigen-presenting cells or lipopolysaccharide (LPS) in the presence or absence of heat-treated LH2171 cells and other bacterial strains for comparison. After incubation, proliferation, cytokine production, and cell viability of the immune cells were determined. The LH2171 significantly inhibited the proliferation of MLN immune cells stimulated with anti-CD3/CD28 compared with other bacterial strains. The antiproliferative potency of LH2171 was effective not only on MLN but also on PP and SP stimulated with anti-CD3/CD28 or LPS. The LH2171 also decreased LPS-stimulated IL-6 production from MLN, PP, and SP, and IL-1ß production from SP, but LH2171 did not affect the viability of immune cells. The LH2171 inhibited immune cell proliferation and proinflammatory cytokine (IL-6 and IL-1ß) production. The inhibitory actions were not due to cytotoxicity to immune cells, suggesting that LH2171 is a dairy Lactobacillus strain with beneficial immunoregulatory properties.

MBL1 genotypes in wild boar populations from Sweden, Austria, the Czech Republic, and Japan.

The single nucleotide polymorphism (SNP) G949T in the mannose-binding lectin ( MBL ) 1 gene has been associated with low MBL-A concentration in serum and detected at different frequencies in various European pig populations. However, the origin of this SNP is not known. Part of the MBL1 gene was sequenced in 12 wild boar/Large White crossbred pigs from the second backcross (BC 2 ) generation in a family material originating from two wild boar x Large White intercrosses. Also, MBL-A serum concentration was measured in the entire BC 2 generation (n = 45). Furthermore, the genotypes of 68 wild boars from Sweden, Austria, the Czech Republic, and Japan were determined in regard to five previously described SNPs in MBL1 . The T allele of G949T was present among the BC 2 animals. MBL-A serum concentration in the BC 2 animals showed a bimodal distribution, with one-third of the animals at levels between 0.7 and 1.6 µg mL(-1) and the remaining pigs at levels around 13 µg mL(-1) . There was a co-variation between the presence of the T allele and low MBL-A concentration in serum. The genotyping of the wild boars revealed differences between populations. The T allele of G949T was not detected in the Austrian and Japanese samples and is thus unlikely to be an original feature of wild boars. In contrast, it was present at high frequency (0.35) among the Swedish wild boars, probably representing a founder effect. Five MBL1 haplotypes were resolved. Only two of these were present among the Japanese wild boars compared to four in each of the European populations. This difference may reflect differences in selection pressure and population history.

Structural analysis of MHC alleles in an RSV tumour regression chicken using a BAC library.

The chicken major histocompatibility complex (MHC-B locus) has a strong association with resistance and susceptibility to numerous diseases. We have found a B haplotype designated WLA that associated with the regression of tumours caused by Rous sarcoma virus J strain (RSV-J). Haplotype WLA was identical to the regressive B6 haplotype when partial genotyping was performed (Poultry Science, 89, 2010, 651). We then constructed a bacterial artificial chromosome (BAC) library from a WLA homozygote chicken to evaluate the structure of this regression haplotype and compared it to those of the B6 haplotype. Comparison between WLA and B6 above 59 kb within the 167 kb, including 14 genes from BG1 to BF2, revealed 75 SNPs and 14 indels. However, several genes were identical between WLA and B6, including the BF1 and BF2 genes, which encode a class I molecule previously suggested to be related to the regression phenotype. The BLB2 gene encoding the MHC class II beta chain showed the greatest diversity, with 19 non-synonymous SNPs. A comparison of WLA and B6 haplotpyes that are associated with tumour regression and RIRa and B24 haplotypes associated with tumour progression suggests that DMA1, DMA2, BRD2, TAPBP and BLB2 genes are not involved in the intensity of RSV J tumour regression.

Changes in gene expression in a porcine preadipocyte cell line during differentiation.

Adipocyte differentiation plays an important role in the formation of fat tissues in pigs and affects meat quality and productivity. Clarification of the nature of the pig genes that participate in adipocyte differentiation will provide a clue to the regulation of fat content and thickness in pig carcases by dietary control; it will also help to find target genes for exploring potentially useful polymorphisms for molecular breeding aimed at fat traits. We constructed a DNA oligomer microarray based on pig transcripts, and we used the array to investigate time-dependent changes in gene expression in the PSPA porcine preadipocyte cell line during differentiation into adipocytes. We selected genes with markedly altered expression (at least fivefold difference in comparison with expression in undifferentiated cells) and classified them into five groups according to gene expression pattern. In the early stage after stimulation of adipocyte differentiation, we observed up-regulation of many genes encoding proteins involved in regulating cell proliferation and transcription. Among the probes corresponding to transcripts that showed marked changes in expression, 27 were located within previously reported QTL regions for traits related to adipose tissues. These results will be valuable resources for finding the genes responsible for fat-related traits that have been identified in previous studies using various pig resource families.

A genome-wide scan for quantitative trait loci affecting respiratory disease and immune capacity in Landrace pigs.

Respiratory disease is the most important health concern for the swine industry. Genetic improvement for disease resistance is challenging because of the difficulty in obtaining good phenotypes related with disease resistance; however, identification of genes or markers associated with disease resistance can help in the genetic improvement of pig health. The purpose of our study was to investigate whether quantitative trait loci (QTL) associated with disease resistance were segregated in a purebred population of Landrace pigs that had been selected for meat production traits and mycoplasmal pneumonia of swine (MPS) scores over five generations. We analysed 1395 pigs from the base to the fifth generation of this population. Two respiratory disease traits [MPS scores and atrophic rhinitis (AR) scores] and 11 immune-capacity traits were measured in 630-1332 animals at 7 weeks of age and when the animal's body weight reached 105 kg. Each of the pigs, except sires in the base population, was genotyped using 109 microsatellite markers, and then, QTL analysis of the full-sib family population with a multi-generational pedigree structure was performed. Variance component analysis was used to detect QTL associated with MPS or AR scores, and the logarithm of odds (LOD) score and genotypic heritability of the QTL were estimated. Five significant (LOD > 2.51) and 18 suggestive (LOD > 1.35) QTL for respiratory disease traits and immune-capacity traits were detected. The significant QTL for Log-MPS score, located on S. scrofa chromosome 2, could explain 87% of the genetic variance of this score in this analysis. This is the first report of QTL associated with respiratory disease lesions.

SLA-DRB1 and -DQB1 genotyping by the PCR-SSOP-Luminex method.

A simple and novel genotyping method was developed to detect alleles at the swine leukocyte antigen (SLA)-DRB1 and -DQB1 class II loci by using polymerase chain reaction (PCR)-fluorescently labeled sequence-specific oligonucleotide probes (SSOPs) and Luminex 100 xMAP detection. The PCR-SSOP-Luminex method exhibited accuracy of 95% for both SLA-DRB1 and -DQB1 in 6 homozygous and 16 heterozygous pig samples as confirmed by sequencing the PCR products of the same samples. In addition, 12 low-resolution SLA class II haplotypes consisting of 7 and 9 DRB1 and DQB1 alleles were identified, respectively, in one population of 283 Landrace pigs. This genotyping method facilitates the rapid and accurate identification of two- or four-digit alleles at the SLA-DRB1 and -DQB1 loci.

Gene cluster for biosynthesis of thermophilin 1277--a lantibiotic produced by Streptococcus thermophilus SBT1277, and heterologous expression of TepI, a novel immunity peptide.

AIMS: To identify genes cluster for thermophilin 1277 produced by Streptococcus thermophilus SBT1277. METHODS AND RESULTS: To identify genes for thermophilin 1277 production, the chromosomal DNA region surrounding the structural gene, tepA, was sequenced using a primer-walking method. The thermophilin 1277 biosynthesis gene locus (tep) is a 9·9-kb region, which consists of at least ten open reading frames (ORFs) in the following order: tepAMTFEGKRI and ORF4. Homology analysis showed high similarity to genes involved in bovicin HJ50 production by Streptococcus bovis HJ50. tepI encodes a novel, small, positively charged hydrophobic peptide of 52 amino acids, which contains a putative transmembrane segment. By heterologous expression in Lactococcus lactis ssp. cremoris MG1363, the TepI-expressing strain exhibited at least 1·3 times higher resistance to thermophilin 1277. CONCLUSIONS: Thermophilin 1277 biosynthesis genes were encoded by a 9·9-kbp region containing at least ten ORFs. TepI is a novel immunity peptide, which protected Strep. thermophilus SBT1277 against thermophilin 1277 in addition to TepFEG, a putative ABC transporter. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report regarding a lantibiotic gene cluster produced by Strep. thermophilus strain.

Sequence polymorphisms in porcine homologs of murine coat colour-related genes.

Herein, we report the variability among 57 porcine homologs of murine coat colour-related genes. We identified single nucleotide polymorphisms (SNPs) and insertions/deletions (InDels) within 44 expressed gene sequences by aligning eight pig complementary DNA (cDNA) samples. The sequence alignment revealed a total of 485 SNPs and 15 InDels. The polymorphisms were then validated by performing matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with reference DNA samples obtained from 384 porcine individuals. Of the 384 individuals, three parents of the experimental F(2) family were included to detect polymorphisms between them for linkage mapping. We also genotyped previously reported polymorphisms of 12 genes, and one SNP each in three genes that were detected by performing a BLAST search of the Trace database. A total of 211 SNPs and three InDels were successfully genotyped from our porcine DNA panel. We detected SNPs in 33 of the 44 genes among the parents of an experimental F(2) family and then constructed a linkage map of the 33 genes for this family. The linkage assignment of each gene to the porcine chromosomes was consistent with the location of the BAC clone in the porcine genome and the corresponding gene sequence. We confirmed complete substitutions of EDNRB and MLPH in the Jinhua and Clawn miniature breeds, respectively. Furthermore, we identified polymorphic alleles exclusive to each pig group: 13 for Jinhua, two for Duroc, three for Meishan, four for the Japanese wild boar, one for the Clawn miniature pig and four for the Potbelly pig.

Genotypes of chicken major histocompatibility complex B locus associated with regression of Rous sarcoma virus J-strain tumors.

The chicken MHC-B locus affects the response to several strains of Rous sarcoma virus (RSV). We evaluated the association between haplotypes of the MHC-B locus and responses to the J strain of RSV by using an F(2) experimental resource family constructed with tumor-regressive (White Leghorn) and tumor-progressive (Rhode Island Red) chickens. The MHC-B haplotypes were determined by genotyping of the microsatellite marker LEI0258 and MHC-B locus class I alpha chain 2 (BF2). Two haplotypes in the resource family, one associated with tumor regression and one with progression, were defined by these 2 markers. To discriminate more precisely the regressive haplotype in this family, we further developed 35 SNP markers at the MHC-B locus. Information on the haplotypes revealed here should be useful for identifying chickens with regression and progression phenotypes of J-strain RSV-induced tumors.

A unique lantibiotic, thermophilin 1277, containing a disulfide bridge and two thioether bridges.

AIMS: To identify the chemical structure of a bacteriocin, thermophilin 1277, produced by Streptococcus thermophilus SBT1277. METHODS AND RESULTS: Thermophilin 1277 was purified and partial N-terminal sequence analysis revealed 6 unidentified amino acids amongst 31 amino acids residues. A 2.7-kbp region containing the thermophilin 1277 structural gene (tepA) encoding 58 amino acids was cloned and sequenced. Mature thermophilin 1277 (33 amino acids) was preceded by a 25-amino acid putative leader peptide containing a double glycine cleavage motif. Peptide sequence analysis following chemical modification of thermophilin 1277 revealed that the Cys21 and Cys29 residues form a disulfide bridge and that Thr8 or Thr10 forms two 3-methyllanthionines with Cys13 or Cys32 via thioether bridges. Antimicrobial activity was disrupted by ethanethiol or reductive agent treatments, indicating that the internal amino acid modifications are crucial for the activity. CONCLUSIONS: Thermophilin 1277 from Strep. thermophilus SBT1277 belongs to the class of AII-type lantibiotics that has a disulfide and two thioether bridges. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of a lantibiotic produced by a GRAS species of Strep. thermophilus; thermophilin 1277 has a unique structure containing both a disulfide bridge and two thioether bridges that are crucial for its activity.

Analysis of the expression of porcine CD200R1 and CD200R1L by using newly developed monoclonal antibodies.

CD200R1 and CD200R1-like are paired receptors which modulate activation of immune cells. Here, we describe the characterisation of their porcine homologues. Analysis of database porcine sequences shows an exceptionally high homology between the extracellular Ig-like domains of these receptors, being the rest more dissimilar. We have obtained two mAbs, PCT1 and PCT3, against a CD200R1-Fc recombinant protein, that bind on CHO cells expressing GFP-tagged CD200R1. The specificity of these mAbs was analysed on CD200R1 L, and also on a CD200R1 splicing variant that lacks the V-type Ig domain. PCT1 bound to both CD200R1 and CD200R1L, but not to the splicing variant, what suggests that recognises an epitope in the V-type Ig domain. PCT3 reacted with both CD200R1 variants, but not CD200R1L, probably binding to an epitope in the N-terminal sequence of CD200R1. Analysis of porcine cells with these mAbs showed expression of CD200R1/CD200R1L on B cells, monocytes and alveolar macrophages.

Molecular characterization of porcine Siglec-10 and analysis of its expression in blood and tissues.

Siglecs are sialic acid binding Ig-like proteins involved in the control of leukocyte responses. In this study we describe the characterization of a porcine orthologue of Siglec-10. A cDNA clone was obtained from a porcine library which encodes a protein with sequence homology to human Siglec-10. This cDNA codes for a type I transmembrane protein containing four Ig-like domains, a transmembrane region, and a cytoplasmic tail with three tyrosine-based motifs, including a membrane-proximal Grb2-binding motif, and two ITIM motifs. When expressed on transfected cells, porcine Siglec-10 was able to bind red blood cells in a sialic acid-dependent manner. Monoclonal antibodies were developed against this protein and used to examine its cell and tissue distribution in the pig. Siglec-10 was found to be expressed on blood B cells and B cell areas of the spleen and lymph nodes. A weak expression was also detected on monocytes.

Molecular and functional characterization of porcine Siglec-3/CD33 and analysis of its expression in blood and tissues.

A cDNA clone encoding a 380 a-a type 1 transmembrane protein with homology to human Siglec-3/CD33 was obtained from a swine small intestine library. An analysis of protein sequence identified two immunoglobulin-like domains, a transmembrane region, and a carboxi-terminal tail with two tyrosine-based signalling motifs. Binding assays of Siglec-3 transfected CHO cells to polyacrylamide glycoconjugates showed a preference for α2-6-linked sialic acids. Using mAbs raised against a fragment containing the two Ig-like domains, porcine Siglec-3 was found to be expressed on monocytes and granulocytes, and their bone marrow precursors. It was also detected in lymph node, splenic and alveolar macrophages. MAbs immunoprecipitated, from granulocyte lysates, a protein of 51-60 kDa under both non-reducing and reducing conditions. MAbs were also used to analyse functional activity of Siglec-3 on bone marrow and blood cells. Engagement of Siglec-3 by mAb had no apparent effect on cell proliferation or cytokine production.

Molecular cloning and chromosomal assignment to SSC12p13-->p11 of swine chemokine receptor CCR7.

We cloned a gene encoding the swine chemokine (C-C motif) receptor 7 (CCR7) and clarified its genomic structure and chromosomal assignment. The ORF and deduced amino-acid sequence were highly conserved with human and mouse CCR7. The swine CCR7 gene was mapped to SSC12p13-->p11 by FISH analysis. Stimulation of swine peripheral blood mononuclear cells by IL-12 and IL-18, considered potent inducers of Th1 cells from analyses in humans and mice, downregulated the expression of CCR7. This is the first report of the molecular cloning, chromosomal assignment and characterization of a chemokine receptor in swine.

Construction of a high-resolution comparative gene map between swine chromosome region 6q11-->q21 and human chromosome 19 q-arm by RH mapping of 51 genes.

A comprehensive and comparative map was constructed for the porcine chromosome (SSC) 6q11-->q21 region, where the gene(s) responsible for the maldevelopment of embryos are localized using swine populations of the National Institute of Animal Industry, Japan (NIAI). Since the chromosomal region corresponds to a region of human chromosome (HSA) 19q13.1-->q13.3 based on bi-directional chromosome painting, primer pairs were designed from porcine cDNA sequences identified, on a sequence comparison basis, as being transcripts from genes orthologous to those in the HSA region. Fifty-one genes were successfully assigned to a swine radiation hybrid (RH) map with LOD scores greater than 6. ERF and PSMD8 genes were assigned to SSC4 and SSC1, respectively. The remaining 49 genes were assigned to SSC6, demonstrating that the synteny between the SSC6 and HSA19 chromosomal regions is essentially conserved, therefore confirming, the results of bi-directional chromosome painting. However, when examined precisely, rearrangements have apparently occurred within the region of conserved synteny. For the ERF and PSMD8 genes assigned to SSCs other than SSC6, additional mapping using somatic cell hybrid (SCH) panels was performed to confirm the results of RH-mapping.

Elucidation of correspondence between swine chromosome 4 and human chromosome 1 by assigning 27 genes to the ImpRH map, and development of microsatellites in the proximity of 14 genes.

Loci affecting swine intramuscular fat content, backfat thickness, carcass weight, and daily weight gain were assigned to regions of swine chromosome (SSC) 4, which were shown to correspond to human chromosome (HSA) 1p22--> q25 by ZOO-FISH, bidirectional chromosome painting, as well as by the linkage map of genes. In order to select candidate genes responsible for the above traits from the human genome database, precise correspondence between SSC4 and HSA1 is a prerequisite. In the present study, 27 genes, PTGFR, GBP1, GBP2, GFI1, GCLM, ABCD3, EXTL2, KCNA3, ADORA3, KCND3, WNT2B, NRAS, SYCP1, PTGFRN, IGSF2, NOTCH2, S100A10, SHC1, SSR2, LMNA, CCT3, CD5L, PEA15, FCER1G, EAT2, DDR2, and LAMB3, located in the HSA1 region corresponding to SSC4 or possibly SSC4, were assigned to the IMpRH map. The alignment of genes from centromere to telomere in the SSC4 q arm is basically conserved in HSA1p22-->q25 with the direction from the q arm to the p arm, which is in good agreement with results from linkage mapping. In addition, the present study first demonstrated that WNT2B residing in the middle of the HSA1 region was assigned to SSC18 with a high lod score (> 5), and that at least three intrachromosomal rearrangements occurred in the region in the process of swine and human evolution. PTGFR, and LAMB3 localized at both ends of the HSA1 region were assigned to SSC6 and SSC9, respectively, which is consistent with regional correspondence reported earlier. In the course of the above analysis, microsatellite markers were developed in the proximity of eleven genes localized on SSC4, and three genes on other swine chromosomes.

Overlapping epitopes of friend murine leukemia virus gag-encoded leader sequence recognized by single cytotoxic T-lymphocyte clones.

The leader signal sequence of the non-structural gag-encoded glycoprotein precursor, Pr75gag, of Friend murine leukemia virus (F-MuLV) contains overlapping epitopes, SIVLCCLCL (p71-79) and CCLCLTVFL (p75 83) that activate Friend virus (FV)-induced tumor (FBL-3)-specific cytotoxic T-lymphocytes (CTL) (Kondo et al., J. Virol., 69, 1995, 6735-6741; Chen et al., J. Virol., 70, 1996, 7773-7782). It was investigated whether these two peptides are recognized by a single CTL clone or by individual clones with different specificities. The results show that both hydrophobic and cysteine-containing peptides are bound to H-2Db class I major histocompatibility complex (MHC) molecules and cross-recognized by a single CTL clone as well as bulk-cultured CTL from the spleens of mice immunized with FBL-3. The peptide p71-79 was effective for sensitizing target cells to lysis by CTL in the concentration of common antigenic peptides. Moreover, peptide p75-83 was 1000-fold more potent than the peptide p71-79. Specific cytotoxicity assays with variant peptides with alanine- and serine-substitutions suggested a highly complex function of the disulfide bond-forming peptides potentially sensitive to small sequence differences. The dominance of CTL responses to the transmembrane region is discussed in light of the high affinity of a novel hydrophobic peptide to compete with other peptides for binding to MHC molecules.