Motoaki Kosugiyama

Polymerase chain reaction assay for detection of sheep and goat meats

Polymerase Chain Reaction (PCR) was applied to a qualitative differentiation between sheep, goat and bovine meats. Oligonucleotide primers were designed for the amplification of sheep satellite I DNA sequence. The PCR amplified 374 bp fragments from sheep and goat DNA, but no fragment from bovine, water buffalo, sika deer, pig, horse, rabbit and chicken DNA. Sheep DNA (10 pg) was detected by 4% agarose gel electrophoresis following PCR amplification. Althoug cooking of the sample meats reduced the PCR products, sheep DNA was detected in the meat heated at 120°C. In order to differentiate between sheep and goat meats, nucleotide sequences of the PCR products were determined directly by cycle sequencing. The sequence of PCR products showed 92% of homology between sheep and goat. They were differentiated by ApaI digestion of the PCR products because sheep had one ApaI site and goat had no site in the PCR products.Polymerase Chain Reaction (PCR) was applied to a qualitative differentiation between sheep, goat and bovine meats. Oligonucleotide primers were designed for the amplification of sheep satellite I DNA sequence. The PCR amplified 374 bp fragments from sheep and goat DNA, but no fragment from bovine, water buffalo, sika deer, pig, horse, rabbit and chicken DNA. Sheep DNA (10 pg) was detected by 4% agarose gel electrophoresis following PCR amplification. Althoug cooking of the sample meats reduced the PCR products, sheep DNA was detected in the meat heated at 120°C. In order to differentiate between sheep and goat meats, nucleotide sequences of the PCR products were determined directly by cycle sequencing. The sequence of PCR products showed 92% of homology between sheep and goat. They were differentiated by ApaI digestion of the PCR products because sheep had one ApaI site and goat had no site in the PCR products.

Molecular phylogeny based on the κ-casein and cytochrome b sequences in the mammalian suborder Ruminantia

Nucleotide sequences for the κ-casein precursor proteins have been determined from the genomic DNAs or hair roots of the Ruminantia. The coding regions, exons 2, 3, and 4, were amplified separately via the three kinds of PCRs and then directly sequenced. The primers were designed from the sequence of bovine κ-casein gene; they were applicable for the amplification of the κ-casein genes from the 13 species in the Ruminantia except exon 2 of the lesser mouse deer. These results permitted an easy phylogenetic analysis based on the sequences of an autosomal gene. A phylogenetic tree was constructed from the mature K-casein sequences and compared with the tree of the cytochrome b genes which were sequenced from the same individuals. The Cervidae (sika deer, Cervus nippon) were separated from the branch of the Bovidae on the tree of κ-casein genes with a relatively high confidence level of the bootstrap analysis, but included in the branch of the Bovidae on the tree of cytochrome b genes. The κ-casein tree indicated a monophyly of the subfamily Caprinae, although the internal branches were uncertain in the Caprinae. The tree based on the nucleotide sequences of cytochrome b genes clearly showed the relationships of the closely related species in the genus Capricornis consisting of serow (C. smatorensis), Japanese serow (C. crispus), and Formosan serow (C. swinhoei). These results would be explained by the difference of resolving power between the κ-casein and the cytochrome b sequences.

Association of BoLA-DRB3 alleles identified by a sequence-based typing method with mastitis pathogens in Japanese Holstein cows.

The association of the polymorphism of bovine leukocyte antigen (BoLA-DRB3) genes identified by the polymerase chain reaction sequence-based typing (PCR-SBT) method with resistance and susceptibility to mastitis caused by pathogenic bacteria was investigated. Blood samples for DNA extraction were collected from 194 Holstein cows (41 healthy cows and 153 mastitis cows including 24 mixed-infection cows infected with 2 or 3 species of pathogens) from 5 districts of Chiba prefecture, Japan. Sixteen BoLA-DRB3 alleles were detected. The 4 main alleles of DRB3*0101, *1501, *1201, and *1101 constituted 56.8% of the total number of alleles detected. Mastitis cows were divided into 2 groups: group 1 with single-infection cows and group 2 with all mastitis cows including 24 mixed-infection cows. The differences in the frequencies of BoLA-DRB3 alleles and the number of cows homozygous or heterozygous for each BoLA-DRB3 allele between healthy cows and the 2 groups of mastitis cows were evaluated. Furthermore, similar comparisons were performed between healthy cows and the 2 groups of mastitis cows for each mastitis pathogen. It was considered that the 4 alleles, namely, DRB3*0101, *1501, *1201, and *1101 had specific resistance and susceptibility to 4 different mastitis pathogens. Thus, DRB3*0101 might be associated with susceptibility to coagulase-negative Staphylococci and Escherichia coli, and DRB3*1501 might be associated with susceptibility to Escherichia coli. However, DRB3*1101 might be associated with resistance to Streptococci and coagulase-negative Staphylococci, and DRB3*1201, with resistance to Streptococci, Escherichia coli, and Staphylococcus aureus.

Association of the amino acid motifs of BoLA-DRB3 alleles with mastitis pathogens in Japanese Holstein cows.

The association of the polymorphism of bovine leukocyte antigen (BoLA-DRB3) genes, identified by the polymerase chain reaction sequence-based typing (PCR-SBT) method, with resistance and susceptibility to mastitis caused by Streptococci, coagulase-negative Staphylococci, Escherichia coli and Staphylococcus aureus was investigated. Blood samples for DNA extraction were collected from 170 Holstein cows (129 mastitis and 41 healthy cows) from 5 districts in Chiba prefecture, Japan. Susceptibility or resistance to the mastitis-causing pathogens was thought to vary by the presence of amino acid substitutions at the 9, 11, 13, and 30 positions. DRB3*0101 and DRB3*1501 had amino acid motifs of Glu(9), Ser(11), Ser(13), and Tyr(30), and they were considered to have susceptibility to all 4 mastitis pathogens. In contrast, DRB3*1101 and DRB3*1401 had amino acid motifs of Gln(9), His(11), Gly(13), and His(30) in these positions, and they also had Val(86), so these alleles were considered to have resistance to Streptococcal and coagulase-negative Staphylococcal mastitis. However, in the case of Escherichia coli mastitis, amino acid substitutions at the 9, 11, 13, and 30 positions had little effect, but rather substitutions at the 47, 67 positions of pocket 7, and at the 71, 74 positions of pocket 4, Tyr(47), Ile(67), Ala(71), and Ala(74), were associated with resistance. This motif was present in DRB3*1201.