Conference Presentation

2014

1.	Genetic Differences among Wild, Laboratory, and Commercial Populations of the Japanese Quail Estimated by Microsatellite DNA Markers Nunome, M.¹⁾, Tadano, R.²⁾, Nakano, M.¹⁾, Miki-Kawahara, R.³⁾, Kono, T.³⁾, Takahashi, S.⁴⁾, Kawashima, T.⁴⁾, Fujiwara, A.⁵⁾, Nirasawa, K.⁶⁾, Mizutani, M.¹⁾ and Matsuda, Y.¹⁾ ¹⁾ Nagoya University ²⁾ Gifu University ³⁾ Tokyo University of Agriculture ⁴⁾ National Institute for Environmental Studies ⁵⁾ Nippon Institute for Biological Science ⁶⁾ NARO Institute of Livestock and Grassland Science The 10th Asia Pacific Poultry Conference 2014, Korea Abstract： The draft genome assembly of the Japanese quail (Coturnix japonica) was deposited in 2013. It enhances the importance of this species as an experimental animal model. However, wild populations of the Japanese quail are threatened with extinction, and their population sizes in east Asia have been declined in decades. Besides, genetic introgression of domestic quails into local wild quail populations is becoming a matter of concern in conservation of this species. In Europe, introgressive hybridization between domestic Japanese quails and wild populations of their related species, the common quail (C. coturnix), has been reported in past years. Thus, assessment of genetic differences between wild populations of Japanese quails and commercial and laboratory quails in Japan can help in proposing an appropriate conservation program of this species. Thus, we examined genetic characteristics of 15 laboratory lines, 57 commercial quails and 21 wild quails using 50 microsatellite DNA markers, which were developed based on the draft genome assembly of this species in our previous study. The fragments of the mitochondrial D loop region (252 bp in length) were also analyzed to infer their breeding history. The mitochondrial D-loop sequences exhibited five haplotypes in wild quails, and two of them were shared with laboratory and commercial quails. A neighbor-joining phylogenetic tree revealed that three unique haplotypes of wild quails separately branched off from basal nodes of the haplotypes of laboratory and commercial quails. This result suggested that a commercial population in Japan and laboratory lines examined in this study were recently developed from wild Japanese quails. Microsatellite markers revealed high levels of genetic differences not only between laboratory lines but also among laboratory, commercial and wild quails. Network analysis based on Fst distances estimated using the microsatellite markers subdivided 15 laboratory lines into two groups, each of which corresponded to quail lines originated in Japan for egg production and quail lines bred in foreign countries for meat. Wild and commercial quails were closely related to the quail lines from foreign countries, especially NIES-Br and NIES-Hn, rather than laboratory lines in Japan. The expected heterozygosities in quail lines from foreign countries (NIES-Br: He = 0.45, NIES-Hn: He = 0.48) are similar to those of commercial quails (He = 0.56) and wild quails (He = 0.46), which were much higher than laboratory quail lines in Japan (e.g. AMRP: He = 0.21, TKP: He = 0.10). These results suggested that quail lines from foreign countries and commercial quails in Japan maintain high genetic variations, and possibly have retained alleles derived from ancestral wild quail populations, which resulted into close relationships of these lines to wild Japanese quails. To obtain more detailed genetic information on wild, commercial, and laboratory Japanese quails, we should study more populations, especially populations of wild and commercial quails, using microsatellite markers rather than mtDNA.
2.	A tentative analysis of genetic differences between wild and laboratory populations of Japanese quails using microsatellite DNA markers Nunome, M.¹⁾, Tadano, R.²⁾, Nakano, M.¹⁾, Miki-Kawahara, R.³⁾, Kono, T.³⁾, Takahashi, S.⁴⁾, Kawashima, T.⁴⁾, Fujiwara, A.⁵⁾, Nirasawa, K.⁶⁾, Mizutani, M.¹⁾ and Matsuda, Y.¹⁾ ¹⁾ Nagoya University ²⁾ Gifu University ³⁾ Tokyo University of Agriculture ⁴⁾ National Institute for Environmental Studies ⁵⁾ Nippon Institute for Biological Science ⁶⁾ NARO Institute of Livestock and Grassland Science International Ornithological Congress 2014, Tokyo Abstract： The draft genome assembly of the Japanese quail (Coturnix japonica) was deposited last year. It enhances the importance of this species as an experimental animal model. However, wild populations of the Japanese quail are threatened with extinction and their population sizes have been declined throughout East Asia in decades. Besides, genetic introgression of domestic quails into local wild quail populations is becoming a matter of concern in conservation of this species. In Europe, introgressive hybridization of domestic Japanese quails and wild populations of their related species, the Common quail (C. coturnix) has been reported in the past years. Thus, genetic assessment for wild populations is an essential approach to constitute an appropriate conservation program of this species. Here, we used 50 microsatellite DNA markers, which were developed based on the draft genome assembly of this species in our previous study, to assess genetic differences between 15 laboratory lines including one commercial line and 21 wild individuals collected from Honshu and Tsushima island of Japanese archipelago, and Liaodong Peninsula of China. The fragment of mitochondrial D loop region (252 bp in length) was also analyzed to infer their breeding history. The mitochondrial D-loop sequences exhibited three major haplotypes in laboratory lines, and two of them were shared with wild quails. A neighbor-joining phylogenetic tree revealed that a few unique haplotypes of wild quails differently branched off from basal nodes of the three haplotypes of laboratory lines. This result suggested different maternal origins of laboratory lines. Microsatellite markers revealed high levels of genetic differences not only among laboratory lines but also between laboratory and wild quail populations. We conclude that these microsatellite markers are more effective for genetic assessment of wild and domestic quail populations than the mitochondrial D loop.
3.	Animal health vaccines (Developing high quality poultry & swine vaccine) Hirata, S.¹⁾, Tsutsumi, N.²⁾ ¹⁾ Nisseiken Co.,Ltd. ²⁾ Nippon Institute for Biological Science Animal Health Conference Shanghai, 2014
4.	Oral rice-based vaccine induces active and passive immunity against　enterotoxigenic E. coli-mediated diarrhea in pigs Takeyama, N.^{1), 2)}, Oroku, K.¹⁾, Tokuhara, D.²⁾, Nagai, S.³⁾, Kiyono, H.²⁾ and Yuki, Y.²⁾ ¹⁾ Nippon Institute for Biological Science ²⁾ Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo ³⁾ Nisseiken Co.,Ltd. International Pig Veterinary Congress (IPVS) 2015 Abstract：【Introduction】Enterotoxigenic Escherichia coli (ETEC) causes severe diarrhea in both neonatal and weaned pigs. Although the risk of mortality has not reported to be high by the single infection with ETEC, multiple infection with different pathogens exacerbates the diarrheic symptoms. The prevalence of ETEC in diarrheic piglets in Japan is second highest after rotavirus (1). Therefore, hygiene measure or vaccination program to reduce risks of ETEC invasion in neonatal and weaning piglets is requisite. We recently developed a rice-based cholera vaccine expressing CTB (MucoRice-CTB) (2, 3). This vaccine has the advantages of being suited to long-term storage without the need for a cold chain (>1.5 y), and delivery of the vaccine antigen is needle- and syringe-free. Cholera toxin B subunit (CTB) has 78% amino acid homology to B subunit of ETEC heat-labile toxin (LT), which is the major cause of diarrhea. Therefore, we selected MucoRice-CTB as a oral vaccine candidate. 【Materials and Methods】One gram of MucoRice-CTB were mixed with feed and orally administered to 2-month-old minipigs or pregnant sows with for four times at 2-week intervals. Serum and intestinal wash fluids were collected from immunized minipigs and CTB/LTB specific IgG or IgA were measured by ELISA. To measure the lactogenic immunity, colostrums and milk were also collected from sows until 2 weeks after fallowing. Neutralizing effects of produced antibodies against LT were confirmed by intestinal loop assay. 4x10⁶ or 4x10⁸ CFU of enterotoxigenic E.coli strain S7 were injected into ileal loop of immunized minipigs and the accumulated fluid in the loop were measured after 16 Hrs of surgery.【Results】All of vaccinated minipigs produced CTB-specific mucosal IgA as well as systemic IgG and IgA. The higher amount of CTB-specific IgG and IgA were also secreted to the colostrums and the secreted antibodies were passively transferred to sera of the progeny. All antibodies were also cross-reacted to LTB. Moreover, water volumes accumulated in the loops of vaccinated minipigs were significantly reduced than that in control minipigs, indicating that MucoRice-CTB can protect pigs from ETEC-mediated diarrhea.【Conclusions and Discussion】MucoRice-CTB, which generated by rice-protein expression system could induce both active and passive immunity against ETEC in pigs by oral route. LT of ETEC acts at mucosal surface. Therefore, toxin specific secretory IgA in the intestine or maternal milk would suit for neutralizing the toxin. Additionally, MucoRice-CTB could feed to animals simply by mixing with food, that it is easily-handed in farms and veterinary clinical site.