Characterization of canine filaggrin: gene structure and protein expression in dog skin

Background – Filaggrin (FLG) is a key protein for skin barrier formation and hydration of the stratum corneum. In humans, a strong association between FLG gene mutations and atopic dermatitis has been reported. Although similar pathogenesis and clinical manifestation have been argued in canine atopic dermatitis, our understanding of canine FLG is limited. Hypothesis/Objectives – The aim of this study was to determine the structure of the canine FLG gene and to raise anti‐dog FLG antibodies, which will be useful to detect FLG protein in dog skin. Methods – The structure of the canine FLG gene was determined by analysing the publicly available canine genome DNA sequence. Polyclonal anti‐dog FLG antibodies were raised based on the canine FLG sequence analysis and used for defining the FLG expression pattern in dog skin by western blotting and immunohistochemistry. Results – Genomic DNA sequence analysis revealed that canine FLG contained four units of repeated sequences corresponding to FLG monomer protein. Western blots probed with anti‐dog FLG monomer detected two bands at 59 and 54 kDa, which were estimated sizes. The results of immunohistochemistry showed that canine FLG was expressed in the stratum granulosum of the epidermis as a granular staining pattern in the cytoplasmic region. Conclusions and clinical importance – This study revealed the unique gene structure of canine FLG that results in production of FLG monomers larger than those of humans or mice. The anti‐dog FLG antibodies raised in this study identified FLG in dog skin. These antibodies will enable us to screen FLG‐deficient dogs with canine atopic dermatitis or ichthyosis.     

Fecal progestagens to detect and monitor pregnancy in captive female cheetahs (Acinonyx jubatus)

The purposes of the present study were to establish a noninvasive monitoring assay of fecal progestagen measurement to detect pregnancy and to identify the components of fecal progestagens in early, middle and late pregnancy in cheetahs. Feces were collected from 7 female cheetahs and analyzed from 30 days before the last copulation to parturition in 9 pregnancies. Blood was collected from one cheetah. Fecal progestagen and serum progesterone concentrations were determined by enzyme immunoassay (EIA). The profiles of the fecal progestagen concentrations were similar to the serum progesterone profile. Fecal progestagen and serum progesterone concentrations remained at the baseline until copulation. In the mean fecal progestagen profile during pregnancy (92.8 ± 0.4 days; from the last copulation to parturition), the concentrations increased 3-4 days after the last copulation and remained high until parturition. To investigate changes in the components of progestagen metabolites in the tripartite periods of gestation, fecal progestagens were analyzed by HPLC-EIA. Marked immunoreactive peaks consistent with 5α-pregnan-3α/β-ol-20-one and 5α-pregnan-3,20-dione and small peaks consistent with 5β-pregnan-3α/β-ol-20-one were detected. There were no distinct difference in the components of progestagens among the first, second and third trimesters of pregnancy. The hormone assay, as an indicator of fecal 5α-reduced pregnanes, is useful for detecting pregnancy and monitoring pregnant luteal activity in cheetahs.     

Protozoan predation of Escherichia coli in hydroponic media of leafy vegetables

Multiple outbreaks of food poisoning associated with fresh vegetable consumptions have occurred in many countries. Numerous reports have described human pathogenic bacteria, such as Escherichia coli O157:H7 and Salmonella spp., that can internalize into fresh vegetables via root or leaf surfaces. While attempting to obtain the threshold concentration of internalization of E. coli inoculated into hydroponic medium during vegetable cultivation, we observed a rapid decrease in E. coli numbers. In the present study, we determined that the rapid decline in E. coli was not due to a physiological change into a viable but non-culturable (VNC) state. The population crash was instead caused by true bacterial death, as the rapid descent was also confirmed by micro-colony fluorescence in situ hybridization, a culture-independent method that can detect VNC cells. We next monitored the number of E. coli inoculated into intact or filter-sterilized hydroponic medium after cultivation of various types of plants. We found that the number of E. coli in intact hydroponic medium decreased markedly, whereas the level in filter-sterilized hydroponic medium was completely unchanged. This result suggests that biotic factors were present that could be eliminated by filtering. Robust predation of E. coli by protozoa (ciliates and flagellates) was observed using fluorescently labeled bacteria incorporated into the hydroponic medium. Finally, morphological identification of flagellates by scanning electron microscopy revealed the presence of a species of Stramenopiles. These findings suggest the importance of protozoa as bacterial feeders in hydroponic systems and hence the use of these organisms as potential control agents of human pathogenic bacteria.     

Establishment of ES cells from inbred strain mice by dual inhibition (2i)

A number of mouse ES cells from inbred strains have been established to date, but efficiency varies across the different strains. The 129 strain mouse is efficient to establish, whereas C57BL/6 and BALB/c strains are not. It is possible that their genetic backgrounds account for the difference in their ability to establish ES cell lines. In this study, we attempted to establish C57BL/6J and BALB/c Cr ES cells by dual inhibition (2i) using two inhibitors (PD0325901 and CHIR99021) of extracellular signal regulated-kinase (ERK) and glycogen synthase kinase-3 (GSK-3), which promote ES cell differentiation. The results revealed that the establishment efficiencies of C57BL/6J and BALB/c Cr ES cells were remarkably increased by 2i. These ES cells stably expressed pluripotent markers and generated high-contribution chimeras with germline transmission. Furthermore, we generated germline chimeras from C57BL/6J ES cells through the method of gene modification. These findings indicate that 2i is a powerful tool for establishing C57BL/6J and BALB/c Cr ES cells with the ability to generate germline chimeras.     

Prevalence of extended-spectrum β-lactamase-producing Escherichia coli and Klebsiella pneumoniae in food-producing animals

To evaluate the diversity of extended-spectrum β-lactamases (ESBL) genes among food-producing animals, 48 isolates of ESBL-producing Escherichia coli isolates were obtained from rectal samples of broilers, layers, beef cattle and pigs, at the slaughterhouse level. ESBL-carrying E. coli were isolated from 60.0% of individual broiler rectal samples, 5.9% of layers, 12.5% of beef cattle and 3% of pigs. One ESBL-producing Klebsiella pneumoniae was isolated from a broiler. The ESBL-positive E. coli isolates from broilers harbored various ESBL genes: bla (SHV-12), bla(CTX-M-2), bla(CTX-M-14), bla(CTX-M-15) and bla(CTX-M-44). The plasmid DNAs were analyzed by restriction patterns. Homogeneous band patterns were yielded in those of K. pneumoniae and E. coli isolates harboring the bla(CTX-M-2) gene from different farms. No genetic relation between the 2 CTX-M-14 ESBL-producing strains was found by pulsed-field gel electrophoresis, although 2 plasmids in these strains, obtained from different broiler farms, were similar to each other. This study provides evidence that the proliferation of CTX-M-producing E. coli is due to the growth of indigenous CTX-M-producing strains and the possible emergence of strains that acquired CTX-M genes by horizontal transfer in different broiler farms. CTX-M-producing coliforms in broilers should be controlled due to the critical importance of cephalosporins and the zoonotic potential of ESBL-producing bacteria.     

Effects of a twin strain of saccharomyces cerevisiae live cells on mixed ruminal microorganism fermentation in vitro

This experiment was designed to investigate the effects of different concentrations (0, 0.33, 0.66, 0.99, and 1.32 g/L) of a twin-strain of Saccharomyces cerevisiae live cells on in vitro mixed ruminal microorganism fermentation of corn starch, soluble potato starch, and sudangrass hay (60.5%, DM basis) plus concentrate mixture (39.5%, DM basis). Ruminal fluid was collected from two dairy cows, mixed with phosphate buffer (1:2), and incubated (30 mL) anaerobically at 38 degrees C for 6 and 24 h with or without yeast supplement, using 200 mg (DM basis) of each substrate. Medium pH, ammonia-N, and numbers of protozoa were unaffected (P = 0.38) by yeast cells in all substrates. Molar proportion of acetate was unchanged (P = 0.56) with cornstarch and soluble potato starch, but increased quadratically (P = 0.02) with hay plus concentrate by treatment. Addition of yeast cells caused a linear increase of total VFA (P = 0.008) in all substrates. Excluding the soluble potato starch, supplementation of S. cerevisiae resulted in a quadratic increase of propionate (P = 0.01), with a quadratic decrease (P = 0.04) of acetate:propionate. When soluble potato starch was used as a substrate, a linear increase (P = 0.006) of the molar proportion of propionate and a quadratic decrease (P = 0.007) in acetate:propionate was observed by treatment. Molar proportion of butyrate was unchanged (P = 0.35) with cornstarch and soluble potato starch, whereas it decreased linearly (P = 0.007) with hay plus concentrate by yeast cell supplementation. When cornstarch and soluble potato starch were used as a substrate, minor VFA were decreased (P = 0.05) by treatment. Accumulation of lactate was linearly decreased by treatment (P = 0.007) in all substrates. During incubation with hay plus concentrate, IVDMD was linearly increased (P = 0.006), whereas production of methane (linear; P = 0.02) and accumulation of hydrogen was decreased (quadratic; P = 0.005) by treatment after 24 h. These results showed that a twin strain of S. cerevisiae live cells stimulated in vitro mixed ruminal microorganism fermentation with decreased lactate, and a small decrease of methane and hydrogen with hay plus concentrate.     

Effect of Japanese horseradish oil on methane production and ruminal fermentation in vitro and in steers

The effects of alpha-cyclodextrin-horseradish oil complex (CD-HR) on methane production and ruminal fermentation were studied in vitro and in steers. In the in vitro study, diluted ruminal fluid (30 mL) was incubated anaerobically at 38 degrees C for 6 h with or without CD-HR, using cornstarch as substrate. The CD-HR was added at various concentrations (0, 0.17, 0.85 and 1.7 g/L). Treatment affected neither the pH of the medium nor the number of protozoa. Total VFA increased in a linear manner (P = 0.02), and NH3-N decreased quadratically (P = 0.04) as the concentration of CD-HR increased from 0.17 g/L to 1.7 g/L. Molar proportions of acetate decreased in a linear manner (P = 0.03), and propionate increased linearly (P = 0.008) with increasing concentrations of CD-HR. Production of methane was inhibited up to 90%, whereas accumulation of dihydrogen was increased 36-fold by 1.7 g/L of CD-HR supplementation relative to controls. The effect of CD-HR on methane production, ruminal fermentation and microbes, and digestibility was further investigated in vivo using four Holstein steers in a crossover design. The CD-HR supplement was mixed into the concentrate portion of a (1.5:1) Sudangrass hay plus concentrate mixture that was fed twice daily to the steers. Ruminal samples were collected 0, 2, and 5 h after the morning feeding. No effects of CD-HR supplementation on ruminal pH (P = 0.63) or protozoal numbers (P = 0.44) were observed. Molar proportion of acetate was decreased (P = 0.04) and propionate was increased (P = 0.005) by CD-HR treatment. Molar proportion of butyrate was increased (P = 0.05) in CD-HR-supplemented steers. Ruminal NH3-N was decreased (P = 0.05) by treatment. Blood plasma glucose concentration was increased (P = 0.02) and urea-N was decreased (P = 0.04) with CD-HR supplementation. Daily DMI was decreased (P = 0.04), and apparent digestibility of DM (P = 0.13), NDF (P = 0.14), and CP tended (P = 0.14) to be increased by treatment. Methane production was decreased (P = 0.03) by 19%, and the number of methanogens was also decreased (P = 0.03). Although N retention (P = 0.11), total viable bacteria (P = 0.15), and sulfate-reducing bacteria (P = 0.17) were not significantly altered by treatment, tendencies for increases were noted with CD-HR supplementation. The number of cellulolytic (P = 0.38) and acetogenic bacteria (P = 0.32) remained unchanged by treatment. These results indicate that CD-HR supplementation can be used to decrease methane production in steers.     

Ratio of rice reflectance for estimating leaf blast severity with a multispectral radiometer

 Rice reflectance was measured to determine the spectral regions most sensitive to leaf blast infection with a multispectral radiometer. As disease severity increased, reflectance also increased in the 400–500 nm (blue), 570–700 nm (red), and 900–2000 nm regions but decreased in the 500–570 nm and 700–900 nm regions. The increased reflectance in the blue and red regions may be attributed to decreased chlorophyll and carotenoid contents in response to the blast infection. The maximum and minimum reflectance differences occurred at 680 nm and 760 nm for the nondiseased and diseased rice, respectively. The spectral location of maximum sensitivity was 675 nm regardless of disease severity. Rice reflectance ratios were evaluated as indicators of leaf blast severity. Two ratios, R550/R675 (reflectance at 550 nm divided by reflectance at 675 nm), and R570/R675 quantified the significant disease severity. These wavelengths were selected based on the sensitivity minima and maxima. The ratios of nondiseased rice plants varied depending on growth stage. The variation in ratios must be considered when they are used to estimate leaf blast severity. Received: April 2, 2002 / Accepted: August 12, 2002