Roles of p38 MAPK, PKC and PI3-K in the signaling pathways of NADPH oxidase activation and phagocytosis in bovine polymorphonuclear leukocytes

AbstractStimulation of bovine polymorphonuclear leukocytes (PMN) with serum-opsonized zymosan (sOZ) induced the activation of p38 mitogen-activated protein kinase (MAPK), protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3-K) and sOZ-induced O2− production was significantly attenuated by their inhibitors (SB203580 for p38 MAPK, GF109203X for PKC and wortmannin for PI3-K). They caused significant attenuation of sOZ-induced phosphorylation of p47phox as well. Flow cytometric analysis, however, revealed that SB203580 and wortmannin attenuated phagocytosis, but GF109203X facilitated it. The results suggest that p38 MAPK and PI3-K participated in both signaling pathways of NADPH oxidase activation (O2− production) and phagocytosis, and PKC participated in the signaling pathway of NADPH oxidase activation alone

A simplified PCR assay for fast and easy mycoplasma mastitis screening in dairy cattle

A simplified polymerase chain reaction (PCR) assay was developed for fast and easy screening of mycoplasma mastitis in dairy cattle. Species of major mycoplasma strains [Mycoplasma (M.) bovis, M. arginini, M. bovigenitalium, M. californicum, M. bovirhinis, M. alkalescens and M. canadense] in cultured milk samples were detected by this simplified PCR-based method as well as a standard PCR technique. The minimum concentration limit for detecting mycoplasma by the simplified PCR was estimated to be about 2.5 × 103 cfu/mL and was similar to that of the standard PCR. We compared the specificity and sensitivity of the simplified PCR to those of a culture method. Out of 1,685 milk samples cultured in mycoplasma broth, the simplified PCR detected Mycoplasma DNA in 152 that were also positive according to the culture assay. The sensitivity and specificity of the simplified PCR were 98.7% and 99.7%, respectively, for detecting mycoplasma in those cultures. The results obtained by the simplified PCR were consistent with ones from standard PCR. This newly developed simplified PCR, which does not require DNA purification, can analyze about 300 cultured samples within 3 h. The results from our study suggest that the simplified PCR can be used for mycoplasma mastitis screening in large-scale dairy farms

Spin-trapping detection of superoxides in polymorphonuclear leukocytes stimulated with serum-opsonized zymosan

To clarify where oxygen radicals are generated in polymorphonuclear leukocytes (PMNs) during phagocytosis, superoxides (O_2^-) from opsonized zymosan (OZ)-stimulated human PMNs were detected by the ESR and spin-trapping methods. PMNs were preactivated with OZ for the indicated periods of time at 37℃. Then a spin-trapping agent, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), was added to them, and they were further incubated for 30sec for ESR observations. The ESR spectra consisted of two components due to the DMPO-OOH and DMPO-OH spin adducts. To clarify where these spin-adducts were present, cells were separated from extracellular fluid by brief centrifugation and resuspended in Hanks' balanced salt solution. ESR examination of two fractions showed that the DMPO-OOH adducts was present in the cell fraction, whereas the DMPO-OH adducts were present in the extracellular fluid. When DMSO was used as a scavenger of hydroxyl radicals (OH), DMPO-CH_3 adducts were observed in the fluid fraction but not in the cell fraction. Both spin adducts were completely abolished by Cu, Zn-SOD but not catalase. These results indicated that O_2^- were produced inside phagosomes of OZ-stimulated PMNs and OH were produced outside them by spontaneous decomposition of the DMPO-OOH adducts

CHROMOSOMAL ANALYSIS OF THE JAPANESE RACCOON DOG BASED ON THE G- AND C-BANDING TECHNIQUES

The chromosomes of two subspecies of the Japanese raccoon dog (Nyctereutes procyonoides viverrinus ; 4 individuals & N. p. albus ; 2 individuals) were analysed using the G- and C-banding techniques. The Japanese raccoon dog had 42 chromosomes in dipliod number and the fundamental arm number (NF, 'nombre fundamental') was 70. The autosomes comprised 13 pairs of meta or submetacentrics and 7 pairs of acrocentrics. The X chromosome was metacentric and intermediate in size between Nos. 12 and 13 while the Y was the smallest acrocentric with a satellite. The G- and C-banding patterns of N.p. albus were similar to those of N.p. viverrinus. The homologous pair of chromosomes could be identified by the G-banding pattern. Most of the chromosomes were C-band positive and contained centromeric heterochromatin. In one male individual (N.p. viverrinus), modal chromosome numbers showed 41,XY because of the elimination of one acrocentric autosome. However, this male did not show any abnormal features except in diploid number

Relationship between milking management practices and milk somatic cell counts on local dairy farms

推奨されている搾乳衛生が体細胞数に及ぼす影響を知るために、北海道十勝地方の1373戸の酪農家を対象に調査を行った。調査方法は、客観的に評価できる項目を獣医師による聞き取り調査で、また客観的に評価困難な項目については乳検検定時に検定員により調査した。統計学的分析はt検定、一元配置分散分析、多変量解析数量化I類を用いて行った。体細胞数に最も影響度の高かった搾乳衛生は、牛体清潔度、乾乳期治療の有無、ポストディッピングの実施時期、過搾乳の有無、ディッパーの種類、搾乳手袋装着の有無、乳房炎軟膏の注入の仕方であった。また、この7つの作業を影響度の高い作業から順に加えて同時実施していくと、平均バルク乳体細胞数（log（X/10^3））は2.21から2.14へ減少し、同時実施しない作業を増やしていくと2.31から2.45へ増加した。同様に影響度の高い作業から順に同時実施していくと、平均体細胞リニアスコアは2.65から2.36に減少し、同時実施しない作業を増やしていくと2.96から3.76に増加した。以上のことから、現在推奨されている搾乳衛生は、体細胞数を低減する上で重要であることが確認できた。また、体細胞数に相関が高い作業の同時実施により体細胞数の低減効果が高いことが明らかになったことから、これらの作業を含めた推奨作業の同時実施が重要であり、特に相関の高かった作業の確実な実施を指導する必要があると考えられた