Development and Validation of KASP Assays for the Genotyping of Racing Performance-Associated Single Nucleotide Polymorphisms in Pigeons.

Pigeon racing's recent upturn in popularity can be attributed in part to the huge prize money involved in these competitions. As such, methods to select pigeons with desirable genetic characteristics for racing or for selective breeding have also been gaining more interest. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for genotyping-specific genes is one of the most commonly used molecular techniques, which can be costly, laborious and time consuming. The present study reports the development of an alternative genotyping method that employs Kompetitive Allele Specific Polymerase Chain Reaction (KASP) technology with specifically designed primers to detect previously reported racing performance-associated polymorphisms within the LDHA, MTYCB, and DRD4 genes. To validate, KASP assays and PCR-RFLP assays results from 107 samples genotyped for each of the genes were compared and the results showed perfect (100%) agreement of both methods. The developed KASP assays present an alternative rapid, reliable, and cost-effective method to identify polymorphisms in pigeons.

Immunogenicity and Protective Activity of Pigeon Circovirus Recombinant Capsid Protein Virus-Like Particles (PiCV rCap-VLPs) in Pigeons (Columba livia) Experimentally Infected with PiCV.

Pigeon circovirus (PiCV) is the most recurrent virus diagnosed in pigeons and is among the major causative agents of young pigeon disease syndrome (YPDS). Due to the lack of an established laboratory protocol for PiCV cultivation, development of prophylaxis is hampered. Alternatively, virus-like particles (VLPs), which closely resemble native viruses but lack the viral genetic material, can be generated using a wide range of expression systems and are shown to have strong immunogenicity. Therefore, the use of VLPs provides a promising prospect for vaccine development. In this study, transfected human embryonic kidney (HEK-293) cells, a mammalian expression system, were used to express the PiCV capsid protein (Cap), which is a major component of PiCV and believed to contain antibody epitopes, to obtain self-assembled VLPs. The VLPs were observed to have a spherical morphology with diameters ranging from 12 to 26 nm. Subcutaneous immunization of pigeons with 100 µg PiCV rCap-VLPs supplemented with water-in-oil-in-water (W/O/W) adjuvant induced specific antibodies against PiCV. Observations of the cytokine expression and T-cell proliferation levels in spleen samples showed significantly higher T-cell proliferation and IFN- γ expression in pigeons immunized with VLPs compared to the controls (p < 0.05). Experimentally infected pigeons that were vaccinated with VLPs also showed no detectable viral titer. The results of the current study demonstrated the potential use of PiCV rCap-VLPs as an effective vaccine candidate against PiCV.

The neutral N-linked glycans of the Basidiomycetous yeasts Pseudozyma antarctica and Malassezia furfur (Subphylum Ustilaginomycotina).

Pseudozyma antarctica and Malassezia furfur are basidiomycetous yeasts under the subphylum Ustilaginomycotina. P. antarctica is a commensal organism found in certain plant species, while M. furfur is associated with several skin diseases of animals including humans. N-linked glycans of P. antarctica and M. furfur were prepared, digested with glycosidases, and structurally analyzed using high performance liquid chromatography (HPLC) and mass spectrometry (MS). Analyses revealed the presence of neutral N-linked glycans ranging in length from Man3GlcNAc2-PA to Man9GlcNAc2-PA. The two species shared the most abundant neutral N-linked glycan: Manα1-2Manα1-6(Manα1-3)Manα1-6(Manα1-2Manα1-2Manα1-3)Manß1-4GlcNAcß1-4GlcNAc (M8A). The second and third most abundant neutral N-linked glycans for P. antarctica were Manα1-2Manα1-6(Manα1-2Manα1-3)Manα1-6(Manα1-2Manα1-2Manα1-3)Manß1-4GlcNAcß1-4GlcNAc (M9A) and Manα1-6(Manα1-3)Manα1-6(Manα1-3)Manß1-4GlcNAcß1-4GlcNAc (M5A), respectively. In the case of M. furfur, Manα1-2Manα1-6(Manα1-3)Manα1-6(Manα1-2Manα1-3)Manß1-4GlcNAcß1-4GlcNAc (M7A) was the second most abundant, while both M8A and M9A were tied for the third most abundant. The presence of putative galactose residues in the hypermannosylated neutral N-linked glycans is also discussed. This report is the first to analyze the neutral N-linked glycans of P. antarctica and M. furfur.

The neutral N-linked glycans of the ustilaginomycete yeast Sympodiomycopsis paphiopedili.

Sympodiomycopsis paphiopedili is a basidiomycetous yeast under the subphylum Ustilaginomycotina and is a commensal organism originally isolated from the nectar of a plant species in Japan. In this study, the neutral N-linked glycans of S. paphiopedili were prepared and structurally analysed using high-performance liquid chromatography (HPLC) and mass spectrometry (MS). Glycosidase digestion analyses were also performed to verify certain glycan linkages. HPLC and MS analyses revealed the presence of neutral N-linked glycans ranging from Man3 GlcNAc2 -PA to Man9 GlcNAc2 -PA in length. The most abundant neutral N-linked glycan structure in this species was found to be the Manα1-2Manα1-6(Manα1-3)Manα1-6(Manα1-2Manα1-2Manα1-3)Manß1-4GlcNAcß1-4GlcNAc (M8A). Moreover, the second and third most abundant neutral N-linked glycan in S. paphiopedili were the Manα1-2Manα1-6(Manα1-2Manα1-3)Manα1-6(Manα1-2Manα1-2Manα1-3)Manß1-4GlcNAcß1-4GlcNAc (M9A) and the Manα1-6(Manα1-3)Manß1-4GlcNAcß1-4GlcNAc (M3B). On the other hand, the effect of the combination of glycoprotein extraction methods (citrate buffer extraction or bead extraction) and the subsequent glycan release methods (hydrazinolysis or PNGase F digestion) on the detection of N-linked glycan peaks was also examined for S. paphiopedili and Saccharomyces cerevisiae in order to avoid under-representation of N-linked glycan structures. High mannose and possible hypermannosylated glycan peaks were detected in all method combinations in S. cerevisiae with the citrate buffer extraction-hydrazinolysis method giving the highest peak yields as compared with the other methods. Here we report the first account of the structural analysis of the neutral N-linked glycan of S. paphiopedili and the comparison of the effect of combinations of glycoprotein extraction methods and glycan release methods with that of the glycan analysis in S. paphiopedili and S. cerevisiae. Copyright © 2017 John Wiley & Sons, Ltd.