Serological distinction of bovine Salmonella carriers from vaccinated and acutely infected cows.

Identification of Salmonella carriers using lipopolysaccharide (LPS) ELISA serology in a Salmonella-infected herd requires distinction of chronically infected cattle from convalescent and vaccinated cows. Cows responding to Salmonella infection and vaccination produce titers to Salmonella LPS that overlap with the lower titers of some Salmonella carriers. The objective of this study was to determine if the LPS antigen specificity of the bovine humoral immune response to Salmonella LPS antigens differs following vaccination and acute and chronic Salmonella infection. The study focused on the nondiscriminatory area of Salmonella ELISA serology, specifically, peak-titered sera from Salmonella bacterin-vaccinated and experimentally infected cows and low-titered sera from Salmonella carriers. The LPS serogroup specificity of the IgG1 and IgG2 response following acute and chronic Salmonella serotype Dublin infection and Salmonella bacterin vaccination was evaluated using 5 Salmonella serogroup (B, D, E1, C3, and C1) LPS ELISA assays. IgG, titers of carriers, vaccinated, and acutely infected cows were predominantly O antigen specific. Similarly, the IgG2 titers of acutely infected cows were also O antigen specific. In contrast, Salmonella carriers produced an IgG2 response to each of the heterologous LPS antigens (B, E1, C3, and C1) examined. The results of this study indicate that the bovine IgG1 isotype response to Salmonella LPS is serogroup specific. Conversely, production of IgG2 antibodies to core Salmonella LPS antigens shared across Salmonella serogroups is a feature of chronic Salmonella infections.     

Molecular and Evolutionary Analysis of the Hd6 Photoperiod Sensitivity Gene Within Genus Oryza

Heading date determines rice’s adaptation to its area and cropping season. We analyzed the molecular evolution of the Hd6 quantitative trait locus for photoperiod sensitivity in a total of 20 cultivated varieties and wild rice species and found 74 polymorphic sites within its coding region (1,002 bp), of which five were nonsynonymous substitutions. Thus, natural mutations and modifications of the coding region of Hd6 within the genus Oryza have been suppressed during its evolution; this is supported by low Ka (≤0.003) and Ka/Ks (≤0.576) values between species, indicating purifying selection for a protein-coding gene. A nonsynonymous substitution detected in the japonica variety “Nipponbare” (a premature stop codon and nonfunctional allele) was found within only some local Japanese japonica varieties, which suggests that this point mutation happened recently, probably after the introduction of Chinese rice to Japan, and is likely involved in rice adaptation to high latitudes. Phylogenetic analysis and genome divergence using the entire Hd6 genomic region confirmed the current taxonomic sections of Oryza and supported the hypothesis of independent domestication of indica and japonica rice.     

Use of PCR-restriction fragment length polymorphism for the identification of zoonotic mycobacteriosis in zebrafish caused by Mycobacterium abscessus and Mycobacterium chelonae

Skin ulcers, scoliosis, and dropsy-like scale edema were observed in laboratory-maintained zebrafish. Affected fish had multifocal granulomas not only in internal organs such as the liver, intestine, genital organs, kidney, muscle, and spleen but also in the fin, epithelium, gills, and sclera of the eyes. Large numbers of acid-fast-rod-shaped bacteria were observed within the necrotic centers of well-demarcated, multifocal granulomas with Gram's stain and Ziehl-Neelson's stain. The size of the Mycobacterium spp. was 1-2 microm x 2-3 microm with a double-layered cell wall, based upon electron-microscopical features. Definitive diagnosis of these outbreaks was obtained by culture on selective media followed by PCR-restriction fragment length polymorphism analysis (PRA) of the rpoB gene for species identification. The amplified 360-bp products of the rpoB gene of mycobacteria isolated from zebrafish were digested with MspI restriction enzyme, which revealed unique band patterns matching those of Mycobacterium abscessus and Mycobacterium chelonae which are responsible for skin and soft tissue infection caused by rapidly growing mycobacteria in humans. This is the first documentation of the precise identification of zoonotic non-tuberculous mycobacteria isolated from laboratory-maintained zebrafish by the PRA of the rpoB gene; this study thus provides a great deal of useful epidemiological information and reduces the likelihood that epizootics will occur.     

In vitro culture conditions using chemically defined media for in vitro matured and intracytoplasmically inseminated porcine oocytes

The present study investigated in vitro culture methods [droplet and Well of the Well (WOW)] using semi-defined and defined media [modified porcine zygote medium (mPZM)] and the additional effects of insulin on in vitro matured and intracytoplasmically inseminated porcine oocytes. In Experiment 1, in vitro matured and intracytoplasmically inseminated porcine oocytes were cultured for 6 days in the following four groups: 1) mPZM-3 (containing bovine serum albumin) + droplet (30 mul), 2) mPZM-3 + WOW, 3) mPZM-4 (containing polyvinyl alcohol) + droplet, and 4) mPZM-4+ WOW. The culture media (mPZM-3 and mPZM-4) and methods (droplet and WOW) did not significantly affect the cleavage rate, but the blastocyst rate of the oocytes cultured in mPZM-3 was significantly (P<0.01) higher than that of mPZM-4 (20.1 and 9.4%, respectively). The blastocyst rates as percentages of the cleaved oocytes (51.8 and 16.9%) and the hatched blastocyst rate as percentages of the number of blastocysts (12.3 and 2.2%) were also significantly (P<0.01) higher in mPZM-3 compared with those in mPZM-4. There was significant interaction (P<0.05) between the two main factors; the effects of the culture media and methods on the rate of hatched blasyocysts as percentages of the blastocysts produced and, the hatched blastocyst rate (20.3%) as percentages of the number of blastocysts produced in mPZM-3 were significantly (P<0.05) higher than in the other groups. In Experiment 2, the additional effects of insulin (100 ng/ml) in mPZM-3 and mPZM-4 media was investigated in the WOW culture system. Insulin addition did not improve cleavage, blastocyst formation, or the number of cells in blastocysts. However, as in Experiment 1, mPZM-3 resulted in a significantly higher blastocyst rate as percentages of the cleaved oocytes than mPZM-4 (33.9 and 18.4%). These results indicate that a chemically defined medium (mPZM-4) needs to be improved to provide more suitable culture conditions for in vitro development of in vitro matured and intracytoplasmically inseminated porcine oocytes. However, the WOW system may be a useful IVC method for blastocyst development of in vitro matured porcine oocytes following ICSI when a semi-defined medium (mPZM-3) is used.     

Phosphate buffer–extractable organic nitrogen as an index of soil‐N availability for sorghum and pearl millet

The availability of soil nitrogen (N) is usually quantified by the amount of mineralized N as determined after several weeks of soil incubation. Various alternative methods using chemical solvents have been developed to extract the available organic N, which is easily mineralized. We compared one such solution, neutral phosphate buffer (NPB), with conventional incubation and 0.01 M–CaCl₂ extraction, as measures of soil N available to two major cereal crops of the semiarid tropics, based on the total N uptake by plants in a pot experiment. Mineralized N had the highest correlation with N uptake by pearl millet (Pennisetum glaucum L., r = 0.979***) and sorghum (Sorghum bicolor [L.] Moench, r = 0.978***). NPB‐extractable N was also highly correlated with N uptake (pearl millet, r = 0.876***; sorghum, r = 0.872***). Only one major peak was detected when NPB extracts were analyzed using size‐exclusion high‐performance liquid chromatography, regardless of soil properties. In addition, the organic N extracted with NPB was characterized by determining the content of peptidoglycan, the main component of bacterial cell walls. Although the characteristics of NPB‐extractable organic N are still unclear, it offers a promising quick assay of available N.     

Clone identification in Japanese flowering cherry (Prunus subgenus Cerasus) cultivars using nuclear SSR markers

Numerous cultivars of Japanese flowering cherry (Prunus subgenus Cerasus) are recognized, but in many cases they are difficult to distinguish morphologically. Therefore, we evaluated the clonal status of 215 designated cultivars using 17 SSR markers. More than half the cultivars were morphologically distinct and had unique genotypes. However, 22 cultivars were found to consist of multiple clones, which probably originate from the chance seedlings, suggesting that their unique characteristics have not been maintained through propagation by grafting alone. We also identified 23 groups consisting of two or more cultivars with identical genotypes. Most members of these groups were putatively synonymously related and morphologically identical. However, some of them were probably derived from bud sport mutants and had distinct morphologies. SSR marker analysis provided useful insights into the clonal status of the examined Japanese flowering cherry cultivars and proved to be a useful tool for cultivar characterization.     

Effect of feeding sweet‐potato condensed distillers solubles on intake and urinary excretion of minerals in Japanese Black steers

Four Japanese Black steers (16 months of age) were assigned to a 4 × 4 Latin square design to investigate the effect of graded levels of sweet‐potato condensed distillers solubles (SCDS) in their diets on intake and urinary excretion of minerals. The four diets consisted of 0%, 10%, 20% and 30% (dry matter (DM) basis) SCDS, with SCDS replacing commercial concentrate (CC). Intake of K, Cl, S, P and Mg increased linearly with increasing SCDS content. Urinary pH increased linearly with increasing dietary SCDS content. SCDS feeding increased urinary K concentrations (linear and quadratic effects). Urinary concentrations of Cl increased linearly with increasing SCDS content. In contrast, urinary concentrations of Mg decreased with increasing SCDS content. Feeding of SCDS did not apparently affect urinary NH₃,P, Na or Ca concentrations. These results suggest that high SCDS feeding is not a risk for crystallization of minerals leading to the formation of magnesium‐phosphate type calculi: although SCDS contains large amounts of P and Mg, high SCDS feeding decreased the Mg concentration and did not affect the P concentration in urine. Additionally, high SCDS feeding had no apparent effects on plasma concentrations of Na, K, Cl, Ca or inorganic P.