Genome-wide detection of changes in allelic frequency in Landrace pigs selected for resistance to mycoplasma pneumonia of swine.

Closed-pig line breeding could change the genetic structure at a genome-wide scale because of the selection in a pig breeding population. We investigated the changes in population structure among generations at a genome-wide scale and the selected loci across the genome by comparing the observed and expected allele frequency changes in mycoplasma pneumonia of swine (MPS)-selected pigs. Eight hundred and seventy-four Landrace pigs, selected for MPS resistance without reducing average daily gain over five generations, had 37,299 single nucleotide polymorphisms (SNPs) and were used for genomic analyses. Regarding population structure, individuals in the first generation were the most widely distributed and then converged into a specific group, as they were selected over five generations. For allele frequency changes, 96 and 14 SNPs had higher allele frequency changes than the 99.9% and 99.99% thresholds of the expected changes, respectively. These SNPs were evenly spread across the genome, and a few of these selected regions overlapped with previously detected quantitative trait loci for MPS and immune-related traits. Our results indicated that the considerable changes in allele frequency were identified in many regions across the genome by closed-pig line breeding based on estimated breeding value.

Nationwide and long-term molecular epidemiologic studies of mumps viruses that circulated in Japan between 1986 and 2017.

In Japan, major mumps outbreaks still occur every 4-5 years because of low mumps vaccine coverage (30-40%) owing to the voluntary immunization program. Herein, to prepare for a regular immunization program, we aimed to reveal the nationwide and long-term molecular epidemiological trends of the mumps virus (MuV) in Japan. Additionally, we performed whole-genome sequencing (WGS) using next-generation sequencing to assess results from conventional genotyping using MuV sequences of the small-hydrophobic (SH) gene. We analyzed 1,064 SH gene sequences from mumps clinical samples and MuV isolates collected from 25 prefectures from 1986 to 2017. The results showed that six genotypes, namely B (110), F (1), G (900), H (3), J (41), and L (9) were identified, and the dominant genotypes changed every decade in Japan since the 1980s. Genotype G has been exclusively circulating since the early 2000s. Seven clades were identified for genotype G using SH sequence-based classification. To verify the results, we performed WGS on 77 representative isolates of genotype G using NGS and phylogenetically analyzed them. Five clades were identified with high bootstrap values and designated as Japanese clade (JPC)-1, -2, -3, -4, -5. JPC-1 and -3 accounted for over 80% of the total genotype G isolates (68.3 and 13.8%, respectively). Of these, JPC-2 and -5, were newly identified clades in Japan through this study. This is the first report describing the nationwide and long-term molecular epidemiology of MuV in Japan. The results provide information about Japanese domestic genotypes, which is essential for evaluating the mumps elimination progress in Japan after the forthcoming introduction of the mumps vaccine into Japan's regular immunization program. Furthermore, the study shows that WGS analysis using NGS is more accurate than results obtained from conventional SH sequence-based classification and is a powerful tool for accurate molecular epidemiology studies.

Genome-wide association studies for production, respiratory disease, and immune-related traits in Landrace pigs.

Identification of a quantitative trait locus (QTL) related to a chronic respiratory disease such as Mycoplasmal pneumonia of swine (MPS) and immune-related traits is important for the genetic improvement of disease resistance in pigs. The objective of this study was to detect a novel QTL for a total of 22 production, respiratory disease, and immune-related traits in Landrace pigs. A total of 874 Landrace purebred pigs, which were selected based on MPS resistance, were genotyped using the Illumina PorcineSNP60 BeadChip. We performed single nucleotide polymorphism (SNP)-based and haplotype-based genome-wide association studies (GWAS) to detect a novel QTL and to evaluate the possibility of a pleiotropic QTL for these traits. SNP-based GWAS detected a total of six significant regions in backfat thickness, ratio of granular leucocytes to lymphatic cells, plasma concentration of cortisol at different ages, and complement alternative pathway activity in serum. The significant region detected by haplotype-based GWAS was overlapped across the region detected by SNP-based GWAS. Most of these detected QTL regions were novel regions with some candidate genes located in them. With regard to a pleiotropic QTL among traits, only three of these detected QTL regions overlapped among traits, and many detected regions independently affected the traits.

Association of swine leukocyte antigen class II haplotypes and immune-related traits in a swine line selected for resistance to mycoplasmal pneumonia.

By selective breeding for five generations, a Landrace line has been recently established to improve resistance to mycoplasmal pneumonia of swine (MPS), daily gain (DG), back fat thickness (BF), and plasma cortisol concentrations (COR). To clarify the involvement of swine leukocyte antigen (SLA) polymorphisms in the selection process, we investigated possible associations of 11 SLA-class II haplotypes with selected traits or immune parameters. Pigs with the low-resolution SLA haplotype Lr-0.23 or Lr-0.13, which increased in frequency with the passage of generations, had less severe pathological lesions of MPS, increased leukocyte phagocytic activity, and higher white blood cell counts. In contrast, Lr-0.12 and Lr-0.2, which decreased in subsequent generations, were weakly associated with more severe pathological lesions of MPS. Therefore, in the studied Landrace line, the Lr-0.23 and Lr-0.13 haplotypes are potentially useful genetic markers for selecting and breeding animals with less severe pathological lesions of MPS.

Correlated response of peripheral blood cytokines with selection for reduced mycoplasma pneumonia of swine lesions in Landrace pigs.

Mycoplasma pneumonia of swine (MPS) is responsible for significant economic losses in the swine industry. We selected Landrace pigs for reduced MPS pulmonary lesions over five generations, and measured concentrations of the following cytokines: interleukin (IL)-10, IL-13, IL-17, tumor necrosis factor (TNF)-α and interferon (IFN)-γ to estimate their correlation with MPS lesions. Sheep red blood cells (SRBC) were injected twice intramuscularly at 70 and 95 kg body weight. Blood serum samples were collected after 1 week of secondary SRBC inoculation and cytokine concentrations were analyzed by ELISA. Genetic parameters and breeding values were estimated. The heritability estimates of IL-10, IL-13, IL-17, TNF-α and IFN-γ were 0.20 ± 0.06, 0.12 ± 0.06, 0.27 ± 0.07, 0.20 ± 0.10 and 0.05 ± 0.03, respectively. Genetic correlations of IL-17 and TNF-α with pulmonary MPS lesions were high (-0.86 ± 0.13 and 0.69 ± 0.29, respectively) and those of IFN-γ and IL-13 with MPS lesions were moderately negative (-0.45). Through selection, the breeding values of IL-17 and IFN-γ increased substantially and those of TNF-α decreased. These results suggest that innate and cellular immunity are more important for the suppression of pulmonary lesions in MPS than humoral-mediated immunity, such as antibody response.

Correlated responses of respiratory disease and immune capacity traits of Landrace pigs selected for Mycoplasmal pneumonia of swine (MPS) lesion.

Five generations of Landrace pigs selected for average daily gain, backfat thickness, Mycoplasmal pneumonia of swine (MPS) lesion score, and plasma cortisol levels, was executed to decrease the MPS lesion score. Genetic parameters and correlated genetic responses for respiratory disease and peripheral blood immune traits were estimated in 1395 Landrace pigs. We estimated the negative genetic correlation of MPS lesion score with phagocytic activity (PA) at 7 weeks of age (-0.67). The breeding values of PA at 7 weeks of age and 105 kg body weight and the correlated selection response of the ratio of granular leukocytes to lymphocytes at 105 kg body weight were significantly increased, and sheep red blood cell-specific antibody production (AP) was significantly decreased in a selection-dependent manner. Increasing of natural immunological indicators (e.g. PA) and decreasing of humoral immunological indicator (e.g. AP) were observed due to genetically decreasing MPS lesion score.

Polymorphisms in pattern recognition receptors and their relationship to infectious disease susceptibility in pigs.

BACKGROUND: Pattern recognition receptors (PRRs), including Toll-like receptors (TLRs), are censoring receptors for molecules derived from bacteria, viruses, and fungi. The PRR system is a prerequisite for proper responses to pathogens, for example by cytokine production, resulting in pathogen eradication. Many cases of polymorphisms in PRR genes affecting the immune response and disease susceptibility are known in humans and mice. METHODS: We surveyed polymorphisms in pig genes encoding PRRs and investigated the relationship between some of the detected polymorphisms and molecular function or disease onset. RESULTS: Nonsynonymous polymorphisms abounded in pig TLR genes, particularly in the region corresponding to the ectodomains of TLRs expressed on the cell surface. Intracellular TLRs such as TLR3, TLR7, and TLR8, and other intracellular PRRs, such as the peptidoglycan receptor NOD2 and viral RNA receptors RIG-I and MDA5, also possessed nonsynonymous polymorphisms. Several of the polymorphisms influenced molecular functions such as ligand recognition. Polymorphisms in the PRR genes may be related to disease susceptibility in pigs: pigs with a particular allele of TLR2 showed an increased tendency to contract pneumonia. CONCLUSIONS: We propose the possibility of pig breeding aimed at disease resistance by the selection of PRR gene alleles that affect pathogen recognition.

[A case of classical tsutsugamushi disease confirmed after an interval of 15 years in Akita Prefecture, Japan].

We report the confirmation of classical tsutsugamushi disease in August 2008. A 17-year-old woman seen for fever and eschar on the back reported having been bitten by an insect nine days earlier while fishing on the Omonogawa river. The suspected culprit was Leptotrombidium akamushi. During convalescence serum IgM and IgG antibody titers rose significantly against the Kato serotype antigen in indirect immunoperoxidase staining. Epidemiology, clinical symptoms and the antibodies detected suggested classical tsutsugamushi disease infection. Such disease transmitted by L. akamushi have not been reported since 1993 in Akita Prefecture. The public should thus be informed about Orientia tsutsugamushi prevention, in case such disease re-care in this area in the future.

Influence of polymorphisms in porcine NOD2 on ligand recognition.

Nucleotide oligomerization domain 2 (NOD2) is a cytosolic pattern recognition receptor (PRR) that responds to muramyldipeptide (MDP), a component of peptidoglycans of gram positive and negative bacteria. NOD2 is involved in the modulation of signaling pathways for other PRRs, such as Toll-like receptors. Polymorphisms in NOD2 may evoke bowel disorders, and human Crohn's disease is significantly correlated with mis-sense insertion of the NOD2 gene. Such polymorphisms affecting ligand recognition in the NOD2 gene may also influence bowel flora in livestock, which is compromised by bowel diseases such as diarrhea. We investigated the functional variance of mis-sense polymorphisms in ligand recognition by porcine NOD2. The 1949T>C polymorphism, located in the region encoding the hinge domain of the molecule, notably diminished the functional response of porcine NOD2 to MDP. By comparison, the 2197A>C polymorphism, localized in the region corresponding to leucine-rich repeats, significantly augmented the response of porcine NOD2 to the ligand. The 1949C allele was rare among pig breeds, suggesting that this mutation is a disadvantage to pigs in their immune response to microbes. The 2197C allele, in contrast, was widely distributed among Western breeds and is most likely to be derived from wild boars in Asia. This is the first report of a causal relationship between molecular function and polymorphisms in PRRs in non-primate, non-rodent mammals. These findings suggest that the 2197C allele might confer an immune response advantage in modern pig breeds and may be a useful marker for breeding aimed at disease resistance in pigs.

Differences in distribution of single nucleotide polymorphisms among intracellular pattern recognition receptors in pigs.

Pathogens localized extracellularly or incorporated into endosomes are recognized mainly by Toll-like receptors, whereas pathogens and pathogen-derived molecules that invade into the cytoplasm of host cells typically are recognized by intracellular pattern recognition receptors (PRRs), such as retinoic acid-inducible gene (RIG)-like helicases (RLHs) and nucleotide-binding oligmerization domain (NOD)-like receptors (NLRs). RIG-I and melanoma differentiation-associated gene 5 (MDA5), which belong to the RLH family, recognize viral genomic RNA, whereas NOD2, a member of the NLR family, responds to microbial peptidoglycans. These receptors may play an important role in pig opportunistic infectious diseases, such as pneumonia and diarrhea, which markedly impair livestock productivity, such that polymorphisms of these receptor genes are potential targets of pig breeding to increase disease resistance. Here, we report single nucleotide polymorphisms (SNPs) in porcine DDX58, IFIH1, and NOD2, which encode RIG-I, MDA5, and NOD2, respectively. Interestingly, compared with DDX58 and IFIH1, NOD2 abounded in nonsynonymous SNPs both throughout the coding sequence and in sequences encoding domains important for ligand recognition, such as helicase domains for RIG-I and MDA5 and leucine-rich repeats in NOD2. These differences in the distribution of SNPs in intracellular PRRs may parallel the diversity of their ligands, which include nucleic acids and peptidoglycans.

Outbreak of gastroenteritis due to sapovirus.

An outbreak of gastroenteritis occurred at a kindergarten in Yokote City, Japan, between February 2006 and March 2006. Sapovirus was identified in 19 of 26 stool specimens by reverse transcription-PCR. A high viral shedding pattern was found for this strain, which was shown to be antigenically distinct from other genogroups.

Liver repopulation and long-term function of rat small hepatocyte transplantation as an alternative cell source for hepatocyte transplantation.

Hepatocyte transplantation (HT) is an attractive therapeutic modality for liver disease as an alternative for liver organ transplantation. Primary fresh hepatocytes (FHs) are the exclusive cell source that has been used for clinical HT. However, the use of FHs is limited due to a shortage of donor cells. Small hepatocytes (SHs) are hepatic progenitor cells and can be isolated not only from rodents but also from humans. SHs can proliferate in vitro and express liver functions, although conventional hepatocytes lose them within a short period after culture. SH functions in vivo have never been studied. We therefore investigated HT using SHs to evaluate cell engraftment and function compared to HT using FHs. The donor cell number in the SH group was smaller than that in the FH group at HT. The cell engraftment in the SH group was smaller in the liver and larger in the spleen than in the FH group. The cell engraftment in the liver increased after HT; however, that in the spleen decreased after HT in both groups. HT using SHs supported the serum albumin level in the NAR experiment as well as that using FH, and albumin mRNA was detectable in the recipients' tissues at 12 weeks after HT. In conclusion, HT using SHs showed hepatic repopulation similar to that using FHs. This suggests that both SHs and FHs can repopulate the liver as if they were hepatic stem cells. In addition, HT using SHs supported liver functions such as albumin correction at the same level as that using FHs. These observations strongly support the idea that SHs could be an alternative to primary FHs as a novel cell source for future HT.

Progression and regression of biloma after laparoscopic cholecystectomy evaluated by computed tomography.

The natural course of biloma after laparoscopic cholecystectomy (LC) was successfully followed and evaluated by computed tomography (CT). Biloma is one of the major complications after LC. However, the processes of biloma progression and regression have not yet been reported in detail. We encountered a case of minor leakage of bile juice after LC. Unexpected injury of the Luschka duct (accessory bile duct) was suspected. We examined this patient periodically by CT, which is very reliable and easy to use for postoperative evaluation. Biloma gradually developed within 6 months as we suspected. We successfully detected the presence of the Luschka duct by bilomagraphy. The patient was symptomatically silent for the next 6 months as the size of the biloma spontaneously decreased. Therefore, it is not always necessary to carry out treatment for biloma after LC if the patient has no symptoms. This is the first report that shows the progression and regression of biloma evaluated precisely by CT.

Serum hyaluronate level for predicting subclinical liver dysfunction after hepatectomy.

The serum hyaluronate (HA) level reflects sinusoidal endothelial cell function correlated with liver function. We have reviewed multiple liver function indicators from 37 patients who underwent hepatectomy for various liver diseases. The serum HA level was well correlated with the indocyanine green retention rate at 15 minutes (ICGR15), lectin-cholesterol (LCAT), hepatocyte growth factor (HGF), liver uptake ratio of technetium-99m galactosyl human serum albumin (99mTc-GSA) at 15 minutes (HH15), prealbumin, and hepatic uptake ratio of 99mTc-GSA at 15 minutes (LHL15). In addition, the model for end-stage liver disease (MELD) score at 7 days after operation was well correlated with serum HA, ICGR15, HH15, and LHL15. In patients who showed serum an HA level of = 100 ng/ml before hepatectomy, the MELD score had significantly deteriorated by 7 days after hepatectomy. Of the 20 patients who showed a serum HA level < 100 ng/ml before hepatectomy, 11 had high serum HA after hepatectomy. The bilirubin level 7 days after operation in this group was much higher than that for patients who maintained a serum HA level < 100 ng/ml after hepatectomy. In addition, the serum HGF level before hepatectomy in this group was significantly lower. We concluded that the serum HA level is a reliable indicator when evaluating liver function and predicting liver dysfunction after hepatectomy. Furthermore, patients with a significantly low HGF level who have a normal HA level are susceptible to liver dysfunction after hepatectomy.