Occurrence and survival of coliform bacteria, Escherichia coli and Salmonella in various manure and compost

pp. 865–874
Occurrence and survival of fecal-contamination indicator bacteria (coliform bacteria, Escherichia coli and Salmonella ) in various manure and compost samples collected from 23 composting facilities mostly in Kyushu were investigated by using selective media. Coliform bacteria were detected on desoxycholate agar from 11 (38%) of 29 product samples (15 cow dung manure, 4 poultry manure, 2 biosolid compost and 8 food waste compost) at a range of 10² to 10⁶ cfu g¹ dry matter. From positive samples, 21 isolates of possible coliform bacteria were purified. Among them, species of coliform bacteria ( E. coli , E. vulneria , Pantoea sp. and Buttiauxella agrestis ) were identified whereas isolates of Serratia marcescens , not coliform bacteria, were also obtained, suggesting that careful observation was necessary to avoid false positive counting due to the presence of a red colony of S. marcescens that resembled coliform bacteria. Isolates of E. coli were tested for slide aggregation with a set of antiserum against pathogenic E. coli serotypes and negative reaction was obtained for all the isolates tested. Direct detection of E. coli on Chromocult coliform agar and Salmonella on MLCB agar resulted in none and 2 (17%) of 12 samples tested, respectively. The fate of fecal-contamination indicator bacteria as above was followed during compost production on 7 cases at 6 compost facilities and 4 patterns were observed: fecal-contamination indicator bacteria 1) decreased and finally disappeared, 2) decreased once but re-growth was occurred on products, 3) decreased to some extent but remained in products, 4) was not detected throughout production. These results suggest that some fecal-contamination indicator bacteria may survive compost production and appropriate temperature control would be significant for hygiene control of manure and compost.     

RNAi-mediated Knockdown of Xist Does Not Rescue the Impaired Development of Female Cloned Mouse Embryos

In mice, one of the major epigenetic errors associated with somatic cell nuclear transfer (SCNT) is ectopic expression of Xist during the preimplantation period in both sexes. We found that this aberrant Xist expression could be impeded by deletion of Xist from the putative active X chromosome in donor cells. In male clones, it was also found that prior injection of Xist-specific siRNA could significantly improve the postimplantation development of cloned embryos as a result of a significant repression of Xist at the morula stage. In this study, we examined whether the same knockdown strategy could work as well in female SCNT-derived embryos. Embryos were reconstructed with cumulus cell nuclei and injected with Xist-specific siRNA at 6–7 h after oocyte activation. RNA FISH analysis revealed that siRNA treatment successfully repressed Xist RNA at the morula stage, as shown by the significant decrease in the number of cloud-type Xist signals in the blastomere nuclei. However, blastomeres with different sizes (from “pinpoint” to “cloud”) and numbers of Xist RNA signals remained within single embryos. After implantation, the dysregulated Xist expression was normalized autonomously, as in male clones, to a state of monoallelic expression in both embryonic and extraembryonic tissues. However, at term there was no significant improvement in the survival of the siRNA-injected cloned embryos. Thus, siRNA injection was largely effective in repressing the Xist overexpression in female cloned embryos but failed to rescue them, probably because of an inability to mimic consistent monoallelic Xist expression in these embryos. This could only be achieved in female embryos by applying a gene knockout strategy rather than an siRNA approach.     

Digestive enzyme activities of pancreas and intestinal digesta in streptozotocin-induced diabetic piglets

Six (Landrace × Large White) × Duroc crossbred, castrated weanling piglets were used to evaluate the effect of a streptozotocin (STZ) injection to induce diabetes on the activities of digestive enzymes derived from the pancreas (lipase, amylase, trypsin and chymotrypsin) in the pancreas and intestinal digesta. Fasting plasma glucose after the administration of STZ was maintained at the level of 302–491 mg/dL during this experiment, compared with the level of 89–125 mg/dL in the control group, and they were defined as the STZ‐induced diabetic piglets for about 7 weeks. Although their bodyweight increased in proportion to a quadratic curve (P < 0.0001) during 49 days after the administration of STZ, the growth of the STZ‐induced diabetic piglets was slower compared with the control. The STZ‐injection did not affect the percentage of the pancreas in bodyweight. The administration of STZ in the piglets tended to accelerate the activity of lipase (P = 0.06) and depress the activity of amylase (P = 0.15) or chymotrypsin (P = 0.18), as units/kg bodyweight, in the pancreas. In the case of measurement as units/kg bodyweight, the activities of intestinal digesta in the STZ group showed a tendency to be higher than those in control group, irrespective of the sort of enzymes. In conclusion, STZ‐induced diabetic piglets have a moderate digestive ability and the administration of exogenous digestive enzymes is not necessary when they are used as a diabetic animal model.     

Dental incremental lines in Sika deer (Cervus nippon); polarized light and fluorescence microscopy of ground sections

Periodic growth incremental lines are found universally in dental hard tissues. This periodicity theoretically allows for estimation of age, even in days, which would be useful in studies of wild animals. In the present study, enamel and dentin increments of the sika deer (Cervus nippon) were observed in ground sections with a polarized light microscope, and their periodicity was examined by the use of a chronological labeling method with fluorochromes. Enamel increments occurred at a mean interval of 10.6 (SD=1.5) microm, and mean spacing of dentin increments was 17.3 (SD=1.8) microm. Fluorochromic marking revealed that incremental lines form each day in enamel and almost every second day in dentin. The fluorescence-labeled lines suggest that enamel formation of the first molar is complete by the age of 5 months. Due to its longer interval of incremental lines and longer term of formation, we conclude that dentin is more suitable than enamel for day-age estimation in sika deer. Experimental confirmation of incremental growth periodicity in various species can improve the reliability of use of tooth increments for age estimation and life history reconstruction.     

Anesthesia and acoustic stress-induced intra-uterine growth retardation in mice

Stress interferes with reproduction, adversely influencing implantation and fetal growth, and sometimes even leading to abortion. Here, we attempted to evaluate the early gestational effects of uncomfortable sound on pregnant mice and their offspring. Ten-week-old pregnant Jcl:ICR mice were exposed to sound (100 dB, random frequency between 9-34 kHz) for 8 hours on the 3(rd), 5(th) and 7(th) gestational days (GD). The effects of general anesthesia were also investigated, with or without acoustic stress. All groups were examined on the 18(th) GD for fetal growth. Fetal weight, number of ossified sacrococcygeal vertebrae and placental weight were all significantly reduced (P<0.0001) when stress was induced on the 7(th) GD, but not on the 3(rd) or 5(th) GD. This intra-uterine growth retardation (IUGR) was significantly inhibited by general anesthesia (P<0.0001), although general anesthesia alone induced significant IUGR (P<0.0001) when compared with control mice. This suggests that acoustic exposure indirectly exerts an effect on fetal growth, possibly via a psycho-maternal pathway. We also found that analysis of the number of ossified sacrococcygeal vertebrae is the most sensitive tool for the study of IUGR.     

Significance of mitochondria for porphyrin and heme biosynthesis

It is concluded that mitochondria are involved in three steps of porphyrin and heme biosynthesis-first, in the formation of delta-aminolevulinic acid from glycine and active succinate; second, in the synthesis of protoporphyrin; third, in the incorporation of iron into the porphyrin ring-that is, in heme formation.     

Urinary free metanephrines measurement in dogs with adrenal gland diseases using a new simple liquid chromatography tandem mass spectrometry method

Measurement of urinary metanephrines in spot samples is used for the diagnosis of canine pheochromocytoma (PC). We describe a simple analytical method based on liquid chromatography tandem mass spectrometry (LC-MS/MS) for measuring free metanephrine (MN) and normetanephrine (NMN) in spot urine samples. Using the developed method, we evaluated the stability of urinary free-MN and free-NMN at various storing conditions. In addition, we assessed the feasibility of urinary free-MN and -NMN measurement for diagnosing PC. Urine samples were mixed with stable isotope internal standards and thereafter purified by ultrafiltration. The purified samples were analyzed by LC-MS/MS in the multiple reaction monitoring mode after separation on a multimode octa decyl silyl column. The coefficient of variation of free-MN and -NMN measurement was 7.6% and 5.5%, respectively. The linearity range was 0.5–10 µg/l for both analytes. Degradation was less than 10% for both analytes under any of the storage conditions. The median free-NMN ratio to creatinine of 9 PC dogs (595, range 144–47,961) was significantly higher (P<0.05) than that of 13 dogs with hypercortisolism (125, range 52–224) or 15 healthy dogs (85, range 50–117). The developed method is simple and may not require acidification of spot urine. The results of this preliminary retrospective study suggest that the measurement of urinary free metanephrines is a promising tool for diagnosing canine PC.     

Role of N-acetylglutamate concentration and ornithine transport into mitochondria in urea synthesis of rats given proteins of different quality

The purpose of this study was to find whether the concentration of N-acetylglutamate and ornithine transport into mitochondria would regulate urea synthesis when the dietary protein quality was manipulated. Experiments were done on three groups of rats given diets containing 10 g of gluten, 10 g of casein, or 10 g of whole egg protein/100 g for 10 days. The plasma concentration and urinary excretion of urea, the liver concentration and synthesis of N-acetylglutamate, the liver concentrations of glutamate and lysine, and the liver ornithine transport into mitochondria increased with the decrease in quality of dietary protein. A reverse correlation was observed between the activities of urea cycle enzymes, the plasma concentration of arginine, and urinary excretion of urea under these conditions. N-Acetylglutamate concentration and ornithine transport into mitochondria in the liver were closely correlated with the excretion of urea. These results suggest that greater N-acetylglutamate concentration and ornithine transport into isolated mitochondria in the liver of rats, given the lower quality of protein, stimulate urea synthesis and that the concentrations of glutamate and lysine in the liver are at least partly related to the hepatic N-acetylglutamate synthesis and ornithine transport, respectively.     

Stereochemical course of the generation of 3-mercaptohexanal and 3-mercaptohexanol by beta-lyase-catalyzed cleavage of cysteine conjugates

The product resulting from the reaction between E-2-hexenal and l-cysteine was shown to be a diastereoisomeric mixture of 2-(2-S-l-cysteinylpentyl)-1,3-thiazolidine-4-carboxylic acid 1. Treatment of the conjugate with two sources of cysteine-S-conjugate beta-lyase (tryptophanase from E. coli and a crude enzyme extract prepared from Eubacterium limosum) resulted in the formation of 3-mercaptohexanal. The reaction proceeded with a slight preference for the (S)-configured product, however, with low conversion rate. The role of 3-S-l-cysteinylhexanal 2 as substrate for beta-lyases was demonstrated by in situ generation of 2 from 3-S-(N-acetyl-l-cysteinyl)hexanal using acylase. Opposite enantioselectivity was observed for the liberation of 3-mercaptohexanol from 3-S-l-cysteinylhexanol 5 by the enzyme preparations from Eubacterium limosum and tryptophanase. Various yeasts produced 3-mercaptohexanol starting from 1 as well as from 5. The reactions proceeded without preferential formation of one of the enantiomers.     

Viral nervous necrosis in hatchery-reared larvae and juveniles of Japanese parrotfish, Oplegnathus fasciatus (Temminck & Schlegel)

Abstract. Mass mortalities of hatchery-reared Japanese parrotfish larvae and juveniles, Oplegnathus fasciatus (Temminck & Schlegel), have occurred in Nagasaki Prefecture. Light and electron microscopic examinations showed that the only consistent histopathological feature was extensive nervous necrosis in the spinal cord, spinal ganglia and brain. Numerous non-enveloped virus particles, icosahedral in morphology and measuring about 34 nm in diameter, were found in the cytoplasm of affected neurones and glial cells. Such nervous necrosis is believed to be the major cause of the mass mortalities of hatchery-reared Japanese parrotfish larvae and juveniles.