PGC-based cryobanking, regeneration through germline chimera mating, and CRISPR/Cas9-mediated TYRP1 modification in indigenous Chinese chickens.

Primordial germ cells (PGCs) are vital for producing sperm and eggs and are crucial for conserving chicken germplasm and creating genetically modified chickens. However, efforts to use PGCs for preserving native chicken germplasm and genetic modification via CRISPR/Cas9 are limited. Here we show that we established 289 PGC lines from eight Chinese chicken populations with an 81.6% success rate. We regenerated Piao chickens by repropagating cryopreserved PGCs and transplanting them into recipient chickens, achieving a 12.7% efficiency rate. These regenerated chickens carried mitochondrial DNA from female donor PGC and the rumplessness mutation from both male and female donors. Additionally, we created the TYRP1 (tyrosinase-related protein 1) knockout (KO) PGC lines via CRISPR/Cas9. Transplanting KO cells into male recipients and mating them with wild-type hens produced four TYRP1 KO chickens with brown plumage due to reduced eumelanin production. Our work demonstrates efficient PGC culture, cryopreservation, regeneration, and gene editing in chickens.

In vitro and in vivo development of interspecies Asian elephant embryos reconstructed with pig enucleated oocytes.

Interspecies somatic cell nuclear transfer (iSCNT) has an immense potential to rescue endangered animals and extinct species like mammoths. In this study, we successfully established an Asian elephant's fibroblast cell lines from ear tissues, performed iSCNT with porcine oocytes and evaluated the in vitro and in vivo development of reconstructed embryos. A total of 7780 elephant-pig iSCNT embryos were successfully reconstructed and showed in vitro development with cleavage rate, 4-cell, 8-cell and blastocyst rate of 73.01, 30.48, 5.64, and 4.73%, respectively. The total number of elephant-pig blastocyte cells and diameter of hatched blastocyte was 38.67 and 252.75 µm, respectively. Next, we designed species-specific markers targeting EDNRB, AGRP and TYR genes to verify the genome of reconstructed embryos with donor nucleus/species. The results indicated that 53.2, 60.8, and 60.8% of reconstructed embryos (n = 235) contained elephant genome at 1-cell, 2-cell and 4-cell stages, respectively. However, the percentages decreased to 32.3 and 32.7% at 8-cell and blastocyst stages, respectively. Furthermore, we also evaluated the in vivo development of elephant-pig iSCNT cloned embryos and transferred 2260 reconstructed embryos into two surrogate gilts that successfully became pregnant and a total of 11 (1 and 10) fetuses were surgically recovered after 17 and 19 days of gestation, respectively. The crown-rump length and width of elephant-pig cloned fetuses were smaller than the control group. Unfortunately, none of these fetuses contained elephant genomes, which suggested that elephant embryos failed to develop in vivo. In conclusion, we successfully obtained elephant-pig reconstructed embryos for the first time and these embryos are able to develop to blastocyst, but the in vivo developmental failure needs further investigated.

Clinical features and treatment strategies of febrile urinary tract infection caused by extended-spectrum beta-lactamase-producing Enterobacteriaceae in children: a multicenter retrospective observational study in Japan.

OBJECTIVES: The incidence of infections caused by extended-spectrum beta-lactamase (ESBL)-producing bacteria has increased. This study aimed to clarify the risk factors and treatment strategies for febrile urinary tract infection (fUTI) caused by ESBL-producing bacteria in Japanese children. METHODS: A retrospective observational study was conducted in 21 hospitals among children aged <16 years diagnosed with an fUTI between 2008 and 2017. Clinical data of children with fUTI caused by ESBL-producing and non-ESBL-producing bacteria were compared. RESULTS: Of the 2049 cases of fUTI, 147 (7.2%) were caused by ESBL-producing bacteria. Children in the ESBL group were more likely to have a history of recent antibiotic use or prophylactic antibiotic use, and experience recurrent UTIs (P <0.001) compared with those in the non-ESBL group. Of the 124 cases of fUTI due to ESBL-producing bacteria that were reviewed, 20 and 100 had concordant and discordant antibiotic use, respectively, and four had unknown antibiotic susceptibility. The median time from the start of treatment to fever resolution was 24 hours and did not differ significantly by therapy group (P = 0.39). CONCLUSION: ESBL-producing bacteria should be considered in children with recurrent UTIs and recent antibiotic use. Most children with fUTI experience clinical improvement regardless of the choice of antibiotic.

Uterus-specific transcriptional regulation underlies eggshell pigment production in Japanese quail.

The precursor of heme, protoporphyrin IX (PPIX), accumulates abundantly in the uteri of birds, such as Japanese quail, Coturnix japonica, which has brown-speckled eggshells; however, the molecular basis of PPIX production in the uterus remains largely unknown. Here, we investigated the cause of low PPIX production in a classical Japanese quail mutant exhibiting white eggshells by comparing its gene expression in the uterus with that of the wild type using transcriptome analysis. We also performed genetic linkage analysis to identify the causative genomic region of the white eggshell phenotype. We found that 11 genes, including 5'-aminolevulinate synthase 1 (ALAS1) and hephaestin-like 1 (HEPHL1), were specifically upregulated in the wild-type uterus and downregulated in the mutant. We mapped the 172 kb candidate genomic region on chromosome 6, which contains several genes, including a part of the paired-like homeodomain 3 (PITX3), which encodes a transcription factor. ALAS1, HEPHL1, and PITX3 were expressed in the apical cells of the luminal epithelium and lamina propria cells of the uterine mucosa of the wild-type quail, while their expression levels were downregulated in the cells of the mutant quail. Biochemical analysis using uterine homogenates indicated that the restricted availability of 5'-aminolevulinic acid is the main cause of low PPIX production. These results suggest that uterus-specific transcriptional regulation of heme-biosynthesis-related genes is an evolutionarily acquired mechanism of eggshell pigment production in Japanese quail. Based on these findings, we discussed the molecular basis of PPIX production in the uteri of Japanese quails.

Clinical characteristics of pediatric febrile urinary tract infection in Japan.

BACKGROUND: Febrile urinary tract infection (fUTI) is the most common serious bacterial infection in children. Despite this, there have been no studies examining the clinical features of pediatric fUTI in Japan. The purpose of this study was to describe the clinical characteristics of fUTI in Japanese children. METHODS: A multicenter, retrospective, observational study was conducted at 21 hospitals in Japan. Children under the age of 15 years who were diagnosed with fUTI between 2008 and 2017 were included. The diagnostic criteria were a temperature over 38 °C and the presence of a single bacterial pathogen in urine culture. Patient characteristics were obtained from medical records. RESULTS: In total, 2,049 children were included in the study. The median age was 5 months, and 59.3% were male. It was found that 87.0% of the males and 53.2% of the females were under 1 year of age. The main causative pathogens identified were Escherichia coli and Enterococcus spp., accounting for 76.6% and 9.8% of infections, respectively. CONCLUSIONS: There was a male predominance of fUTI in Japanese children, particularly in infants. Enterococcus spp. were the second most frequent causative pathogen; therefore, Gram staining of urine samples is strongly recommended before initiating antibiotic therapy.

Geographic Origin and Genetic Characteristics of Japanese Indigenous Chickens Inferred from Mitochondrial D-Loop Region and Microsatellite DNA Markers.

Japanese indigenous chickens have a long breeding history, possibly beginning 2000 years ago. Genetic characterization of Japanese indigenous chickens has been performed using mitochondrial D-loop region and microsatellite DNA markers. Their phylogenetic relationships with chickens worldwide and genetic variation within breeds have not yet been examined. In this study, the genetic characteristics of 38 Japanese indigenous chicken breeds were assessed by phylogenetic analyses of mitochondrial D-loop sequences compared with those of indigenous chicken breeds overseas. To evaluate the genetic relationships among Japanese indigenous chicken breeds, a STRUCTURE analysis was conducted using 27 microsatellite DNA markers. D-loop sequences of Japanese indigenous chickens were classified into five major haplogroups, A-E, among 15 haplogroups found in chickens worldwide. The haplogroup composition suggested that Japanese indigenous chickens originated mainly from China, with some originating from Southeast Asia. The STRUCTURE analyses revealed that Japanese indigenous chickens are genetically differentiated from chickens overseas; Japanese indigenous chicken breeds possess distinctive genetic characteristics, and Jidori breeds, which have been reared in various regions of Japan for a long time, are genetically close to each other. These results provide new insights into the history of chickens around Asia in addition to novel genetic data for the conservation of Japanese indigenous chickens.

Transcriptome analysis revealed misregulated gene expression in blastoderms of interspecific chicken and Japanese quail F1 hybrids.

Hybrid incompatibility, such as sterility and inviability, prevents gene flow between closely-related populations as a reproductive isolation barrier. F1 hybrids between chickens and Japanese quail (hereafter, referred to as quail), exhibit a high frequency of developmental arrest at the preprimitive streak stage. To investigate the molecular basis of the developmental arrest at the preprimitive streak stage in chicken-quail F1 hybrid embryos, we investigated chromosomal abnormalities in the hybrid embryos using molecular cytogenetic analysis. In addition, we quantified gene expression in parental species and chicken- and quail-derived allele-specific expression in the hybrids at the early blastoderm and preprimitive streak stages by mRNA sequencing. Subsequently, we compared the directions of change in gene expression, including upregulation, downregulation, or no change, from the early blastoderm stage to the preprimitive streak stage between parental species and their hybrids. Chromosome analysis revealed that the cells of the hybrid embryos contained a fifty-fifty mixture of parental chromosomes, and numerical chromosomal abnormalities were hardly observed in the hybrid cells. Gene expression analysis revealed that a part of the genes that were upregulated from the early blastoderm stage to the preprimitive streak stage in both parental species exhibited no upregulation of both chicken- and quail-derived alleles in the hybrids. GO term enrichment analysis revealed that these misregulated genes are involved in various biological processes, including ribosome-mediated protein synthesis and cell proliferation. Furthermore, the misregulated genes included genes involved in early embryonic development, such as primitive streak formation and gastrulation. These results suggest that numerical chromosomal abnormalities due to a segregation failure does not cause the lethality of chicken-quail hybrid embryos, and that the downregulated expression of the genes that are involved in various biological processes, including translation and primitive streak formation, mainly causes the developmental arrest at the preprimitive streak stage in the hybrids.

The combination of the neutrophil-to-lymphocyte and platelet-to-lymphocyte ratios as a novel predictor of intravenous immunoglobulin resistance in patients with Kawasaki disease: a multicenter study.

INTRODUCTION: The neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) have been reported to be a predictor for intravenous immunoglobulin (IVIG) resistance in patients with Kawasaki disease (KD) recently. The objective of the present study was to elucidate the predictive validity of this new marker in a multicenter study. MATERIALS AND METHODS: We retrospectively reviewed the clinical records of 520 consecutive KD patients (development data set) and 332 subsequent patients (validation data set) at 7 hospitals in Japan. RESULTS: Both NLR and PLR were significantly higher in the IVIG-resistant group than in the IVIG-responsive group. When we set the cut-off point as NLR ≥ 4.11 and PLR ≥ 119, multiple logistic regression analyses showed that a high NLR and PLR before initial IVIG were independent predictors of IVIG resistance, and their combination was a stronger predictor than either alone. The sensitivity and specificity of the combination of NLR ≥ 4.11 and PLR ≥ 119 were 0.58 and 0.73 in the development data set. Validated using an independent data set, they were 0.54 and 0.72 in the validation data set. On comparing the AUC of this predictor with those of the Gunma and Kurume scores, the AUC was highest for this predictor, followed by the Gunma score and Kurume score (0.70, 0.68, and 0.64, respectively). DISCUSSION: The predictive validity of the combination of a high NLR and PLR, which is a simple and convenient indicator, was equal to or better than that of the existing scoring systems. The new predictive marker may be a suitable indicator for predicting IVIG resistance in KD patients.

Combined deletions of IHH and NHEJ1 cause chondrodystrophy and embryonic lethality in the Creeper chicken.

The Creeper (Cp) chicken is characterized by chondrodystrophy in Cp/+ heterozygotes and embryonic lethality in Cp/Cp homozygotes. However, the genes underlying the phenotypes have not been fully known. Here, we show that a 25 kb deletion on chromosome 7, which contains the Indian hedgehog (IHH) and non-homologous end-joining factor 1 (NHEJ1) genes, is responsible for the Cp trait in Japanese bantam chickens. IHH is essential for chondrocyte maturation and is downregulated in the Cp/+ embryos and completely lost in the Cp/Cp embryos. This indicates that chondrodystrophy is caused by the loss of IHH and that chondrocyte maturation is delayed in Cp/+ heterozygotes. The Cp/Cp homozygotes exhibit impaired DNA double-strand break (DSB) repair due to the loss of NHEJ1, resulting in DSB accumulation in the vascular and nervous systems, which leads to apoptosis and early embryonic death.

Genetic diversity of 21 experimental chicken lines with diverse origins and genetic backgrounds.

The genetic characteristics and diversity of 21 experimental chicken lines registered with the National BioResource Project of Japan were examined using mitochondrial D-loop sequences and 54 microsatellite DNA markers. A total of 12 haplotypes were detected in the 500-bp mitochondrial DNA sequences of the hypervariable segment I for 349 individuals of 21 lines. The 12 haplotypes belonged to three (A, D, and E) haplogroups, out of the eight (A‒H) common haplogroups in domestic chickens and red junglefowls. The haplogroups A and D were widely represented in indigenous chickens in the Asian and Pacific regions, and the haplogroup E was the most prevalent in domestic chickens. Genetic clustering by discriminant analysis of principal components with microsatellite markers divided 681 individuals of 21 lines into three groups that consisted of Fayoumi-, European-, and Asian- derived lines. In each of the cladograms constructed with Nei's genetic distances based on allele frequencies and the membership coefficients provided by STRUCTURE and with the genetic distance based on the proportion of shared alleles, the genetic relationships coincided well with the breeding histories of the lines. Microsatellite markers showed remarkably lower genetic heterozygosities (less than 0.1 observed heterozygosity) for eight lines (GSP, GSN/1, YL, PNP, BM-C, WL-G, BL-E, and #413), which have been maintained as closed colonies for more than 40 years (except for #413), indicating their usefulness as experimental chicken lines in laboratory animal science research.

Nonsense mutation in PMEL is associated with yellowish plumage colour phenotype in Japanese quail.

The L strain of Japanese quail exhibits a plumage phenotype that is light yellowish in colour. In this study, we identified a nonsense mutation in the premelanosome protein (PMEL) gene showing complete concordance with the yellowish plumage within a pedigree as well as across strains by genetic linkage analysis of an F2 intercross population using approximately 2,000 single nucleotide polymorphisms (SNPs) that were detected by double digest restriction site-associated DNA sequencing (ddRAD-seq). The yellowish plumage was inherited in an autosomal recessive manner, and the causative mutation was located within an 810-kb genomic region of the LGE22C19W28_E50C23 linkage group (LGE22). This region contained the PMEL gene that is required for the normal melanosome morphogenesis and eumelanin deposition. A nonsense mutation that leads to a marked truncation of the deduced protein was found in PMEL of the mutant. The gene expression level of PMEL decreased substantially in the mutant. Genotypes at the site of the nonsense mutation were fully concordant with plumage colour phenotypes in 196 F2 offspring. The nonsense mutation was not found in several quail strains with non-yellowish plumage. Thus, the yellowish plumage may be caused by the reduced eumelanin content in feathers because of the loss of PMEL function.

The "Extended Brown" Plumage Color Mutant of Blue-Breasted Quail (Coturnix chinensis) is Associated with a Mutation in the Melanocortin 1-Receptor Gene (MC1R).

The mutant plumage color "extended brown (EB)" of the blue-breasted quail was genetically investigated. Mating experiments revealed that the EB plumage is controlled by an autosomal, incompletely dominant allele, for which we propose the symbol Eb. The EB plumage is characterized by dark brown color, and homozygotes for this mutation generally showed darker pigmentation than the heterozygotes. DNA sequencing and PCR-RFLP analyses of the EB mutants showed a rigid association between the EB plumage and a G-to-A nucleotide substitution at position 274 in the melanocortin 1-receptor gene (MC1R), clearly indicating that MC1R is the candidate gene for the EB plumage color in the blue-breasted quail.

Identification of TENP as the Gene Encoding Chicken Egg White Ovoglobulin G2 and Demonstration of Its High Genetic Variability in Chickens.

Ovoglobulin G2 (G2) has long been known as a major protein constituent of chicken egg white. However, little is known about the biochemical properties and biological functions of G2 because the gene encoding G2 has not been identified. Therefore, the identification of the gene encoding G2 and an analysis of its genetic variability is an important step toward the goal of understanding the biological functions of the G2 protein and its utility in poultry production. To identify and characterize the gene encoding G2, we separated G2 from egg white using electrophoresis on a non-denaturing polyacrylamide gel. Two polymorphic forms of G2 protein (G2A and G2B), with different mobilities (fast and slow respectively), were detected by staining. The protein band corresponding to G2B was electro-eluted from the native gel, re-electrophoresed under denaturing conditions and its N-terminal sequence was determined by Edman degradation following transfer onto a membrane. Sequencing of the 47 kDa G2B band revealed it to be identical to TENP (transiently expressed in neural precursors), also known as BPI fold-containing family B, member 2 (BPIFB2), a protein with strong homology to a bacterial permeability-increasing protein family (BPI) in mammals. Full-length chicken TENP cDNA sequences were determined for 78 individuals across 29 chicken breeds, lines, and populations, and consequently eleven non-synonymous substitutions were detected in the coding region. Of the eleven non-synonymous substitutions, A329G leading to Arg110Gln was completely associated with the noted differential electrophoretic mobility of G2. Specifically G2B, with a slower mobility is encoded by A329 (Arg110), whereas G2A, with a faster mobility, is encoded by G329 (Gln110). The sequence data, derived from the coding region, also revealed that the gene encoding G2 demonstrates significant genetic variability across different chicken breeds/lines/populations. These variants, and how they correlate with egg white properties, may allow us to understand further G2's functions.

Embryonic development and inviability phenotype of chicken-Japanese quail F1 hybrids.

Interspecific hybrid incompatibility, including inviability and sterility, is important in speciation; however, its genetic basis remains largely unknown in vertebrates. Crosses between male chickens and female Japanese quails using artificial insemination can generate intergeneric hybrids; however, the hatching rate is low, and hatched hybrids are only sterile males. Hybrid development is arrested frequently during the early embryonic stages, and the sex ratio of living embryos is male-biased. However, the development and sex ratio of hybrid embryos have not been comprehensively analyzed. In the present study, we observed delayed embryonic development of chicken-quail hybrids during the early stage, compared with that of chickens and quails. The survival rate of hybrids decreased markedly during the blastoderm-to-pre-circulation stage and then decreased gradually through the subsequent stages. Hybrid females were observed at more than 10 d of incubation; however, the sex ratio of hybrids became male-biased from 10 d of incubation. Severely malformed embryos were observed frequently in hybrids. These results suggest that developmental arrest occurs at various stages in hybrid embryos, including a sexually non-biased arrest during the early stage and a female-biased arrest during the late stage. We discuss the genetic basis for hybrid inviability and its sex bias.

Chicken Ovotransferrin Variants OTFB and OTFC Harboring Substitution of GAT (Asp) to AAT (Asn) in the Codon 500 and their Antimicrobial Activity.

Chicken egg white ovotransferrin (OTF) has been reported to exist in three electrophoretic variants (OTFA, OTFB and OTFC). In this report, we identified a causal polymorphism between the OTFB and OTFC variants in Japanese and Taiwanese native chickens and compared the antibacterial activity between these two variants. The cDNA sequence analyses from Satsumadori oviducts revealed that three non-synonymous SNPs T1809G (Ser52Ala), A2258G (Ile96Val) and G7823A (Asp500Asn) corresponded to the OTF electrophoretic phenotypes. Of the three SNPs, the G7823A mutation perfectly corresponded to the electrophoretic phenotypes OTFB (G/G, Asp500Asp), OTFB/C (G/A, Asp500Asn) and OTFC (A/A, Asn500Asn) in three chicken populations. The variants OTFB and OTFC exhibited similar antibacterial potency against Gram-positive and Gram-negative bacteria. This study provides, for the first time, molecular information on polymorphism of OTFB and OTFC variants of chicken ovotransferrin and its effect on the antimicrobial activity of the respective variants.

Development of a novel pink-eyed dilution mouse model showing progressive darkening of the eyes and coat hair with aging.

Oca2(p-cas) (oculocutaneous albinism II; pink-eyed dilution castaneus) is a coat color mutant gene on mouse chromosome 7 that arose spontaneously in wild Mus musculus castaneus mice. Mice homozygous for Oca2(p-cas) usually exhibit pink eyes and gray coat hair on the non-agouti genetic background, and this ordinary phenotype remains unchanged throughout life. During breeding of a mixed strain carrying this gene on the C57BL/6J background, we discovered a novel spontaneous mutation that causes darkening of the eyes and coat hair with aging. In this study, we developed a novel mouse model showing this unique phenotype. Gross observations revealed that the pink eyes and gray coat hair of the novel mutant young mice became progressively darker in color by approximately 3 months after birth. Light and transmission-electron microscopic observations revealed a marked increase in melanin pigmentation of coat hair shafts and choroid of the eye in the novel mice compared to that in the ordinary mice. Sequence analysis of Oca2(p-cas) revealed a 4.1-kb deletion involving exons 15 and 16 of its wild-type gene. However, there was no sequence difference between the two types of mutant mice. Mating experiments suggested that the novel mutant phenotype was not inherited in a simple fashion, due to incomplete penetrance. The novel spontaneous mutant mouse is the first example of progressive hair darkening animals and is an essential animal model for understanding of the regulation mechanisms of melanin biosynthesis with aging.

Acute encephalopathy associated with human metapneumovirus infection.

We encountered a 3.5-year-old girl with acute encephalopathy associated with human metapneumovirus (hMPV) infection. She had pyrexia and status epilepticus, followed by a coma. Cerebrospinal fluid analysis showed no pleocytosis or elevation of protein levels. hMPV RNA was detected in tracheal aspirate. Acute encephalopathy in the patient was probably related to the hMPV infection. Serum levels of interleukin-6 and matrix metalloproteinase-9 were elevated on admission, and these factors were presumed to be related to acute encephalopathy, associated with her viral infection, or due to status epilepticus. She was treated with dexamethasone pulse therapy, intravenous immunoglobulin, and continuous thiopental infusion. She recovered without neurological sequelae.

Endothelin receptor B2 (EDNRB2) is responsible for the tyrosinase-independent recessive white (mo(w) ) and mottled (mo) plumage phenotypes in the chicken.

A mutation that confers white plumage with black eyes was identified in the Minohiki breed of Japanese native chicken (Gallus gallus domesticus). The white plumage, with a few partially pigmented feathers, was not associated with the tyrosinase gene, and displayed an autosomal recessive mode of inheritance against the pigmented phenotype. All F1 offspring derived from crosses with mottled chickens (mo/mo), which show characteristic pigmented feathers with white tips, had plumage with a mottled-like pattern. This result indicates that the white plumage mutation is a novel allele at the mo locus; we propose the gene symbol mo(w) for this mutant allele. Furthermore, the F1 hybrid between the mo(w) /mo(w) chicken and the panda (s/s) mutant of Japanese quail (Coturnix japonica), whose causative gene is the endothelin receptor B2 (EDNRB2) gene, showed a mo(w)/mo(w) chicken-like plumage, suggesting the possibility that the mutations in parental species are alleles of the same gene, EDNRB2. Nucleotide sequencing of the entire coding region of EDNRB2 revealed a non-synonymous G1008T substitution, which causes Cys244Phe amino acid substitution in exon 5 (which is part of the extracellular loop between the putative fourth and fifth transmembrane domains of EDNRB2) in the mutant chicken. This Cys244Phe mutation was also present in individuals of four Japanese breeds with white plumage. We also identified a non-synonymous substitution leading to Arg332His substitution that was responsible for the mottled (mo/mo) plumage phenotype. These results suggest that the EDN3 (endothelin 3)-EDNRB2 signaling is essential for normal pigmentation in birds, and that the mutations of EDNRB2 may cause defective binding of the protein with endothelins, which interferes with melanocyte differentiation, proliferation, and migration.

Detection of methicillin-resistant Staphylococcus aureus using a specific anti-PBP2a chicken IgY antibody.

Immunodetection of methicillin-resistant Staphylococcus aureus (MRSA) by conventional methods employing mammalian immunoglobulins has unknown detection limits, and often yields false-positive results because of the presence of S. aureus protein A, which binds the Fc region of mammalian IgG. In this study, a new PBP2a-specific chicken IgY antibody was developed in inbred and conventional chickens, and used for the detection of MRSA using whole cell lysate samples. Our results showed that this chicken IgY antibody minimized the side effects of protein A. Moreover, enzyme-linked immunosorbent assay and immunochromatography systems were used with a monoclonal and polyclonal anti-PBP2a IgY antibody, clearly differentiating MRSA from methicillin-sensitive S. aureus and other methicillin-sensitive Staphylococcus spp. The detection limit of the immunochromatography was 10(8) colony-forming units; therefore, 1 colony on an agar plate was adequate to distinguish MRSA from non-MRSA. The specificity and sensitivity of this assay were almost similar to that of a commercially available latex agglutination test; however, the procedure used in this study was less complicated. The entire detection procedure, including sample preparation, takes only 20 min and does not require special equipment. Therefore, the use of this IgY antibody as a new tool for the detection of MRSA is highly recommended.