Demonstration of Akabane virus antigen using immunohistochemistry in naturally infected newborn calves

Eight newborn calves showing ataxia were necropsied and examined histologically. Six of seven cerebrospinal fluid samples collected from these animals had neutralizing antibody for Akabane virus (AKV). All examined calves had nonsuppurative encephalomyelitis, localized mainly in the midbrain and spinal cord. Corresponding to the encephalitic lesion, AKV antigen was demonstrated in neuroglial cells in the brain stem and neuronal cells in the ventral horn of the spinal cord. This is the first study to demonstrate AKV antigen by immunohistochemistry in naturally infected newborn calves.     

Evidence for transuterine migration of embryos in the domestic cat

A total of 169 pregnant cats presented for ovariohysterectomy were examined for the number of corpora lutea (CL) and the number of fetuses per uterine horn. The implantation rate and the frequency of occurrence of transuterine migration of embryos were calculated. The average number of CL was 5.6 +/- 1.9 (mean +/- SD; range, 2-11), the average number of fetuses was 4.5 +/- 1.4 (range, 1-8) and the average implantation rate was 84 +/- 20%. Transuterine migration occurred in 69/169 cats (40.8%). In animals with a 100% implantation rate, transuterine migration occurred in 50/84 cats (59.5%). The number of embryos that migrated ranged from 1-3 per animal and embryos moved from the uterine horn ipsilateral to the ovary with the larger number of CL in 66/69 cats (95.7%). As a result of transuterine migration, the difference in the number of fetuses between uterine horns as compared with the difference in the number of CL between ovaries was smaller in 54 casts (78.3%), unchanged in 8 cats (11.6%) and larger in 7 cats (10.1%). These results indicate that cats tend to equalize the number of fetuses between uterine horns by transuterine migration of embryos.     

Anti-influenza A virus activities of mannan-binding lectins and bovine conglutinin

Mannan-binding lectin (MBL) and bovine conglutinin (BKg) belong to the collectin family, which is involved in first-line host defense against various infectious agents. We have previously reported that human MBL inhibited type A influenza viral hemagglutination, infection and spreading to adjacent cells without complement activation. In this study, we investigated the direct antiviral activities of bovine MBL, rabbit MBL and BKg. All collectins used in this study inhibited viral infectivity and hemagglutination at concentrations of 0.02-0.3 microg/ml. They also demonstrated inhibitory activity against viral spreading. Like human MBL, bovine MBL and BKg showed antiviral activities at their physiological concentrations. These results suggest that mammalian MBLs and BKg may inhibit the spread of influenza A virus through the bloodstream.     

Tetrodotoxin accumulation conflicts with low salinity tolerance in juvenile tiger puffer Takifugu rubripes

We evaluated whether bearing tetrodotoxin (TTX) affects salinity stress in the juvenile tiger puffer Takifugu rubripes. Juveniles of hatchery-reared non-toxic T. rubripes [body weight (BW): 1.7?±?0.2 g, n?=?120] were divided into six tanks and acclimatized to salinity (8.5 ppt) that is equivalent to blood osmolality. Fish in three tanks were fed non-toxic diet, and those in the other three tanks were fed a TTX-containing diet (356 ng/g diet) three times a day until satiation. In each diet treatment, salinity of one tank was kept at 8.5 ppt, and the other two tanks were adjusted to either 1.7 or 34.0 ppt, and fish were reared for another 33 days. Then, we compared survival, growth, TTX accumulation, plasma osmolality, plasma cortisol, and glucose levels among treatments. We detected TTX only in the fish in the TTX-diet groups. Survival was highest at 8.5 ppt (70%) and lowest at 1.7 ppt in the TTX-diet group (20%). The BW was greater at 8.5 ppt, and plasma osmolality was significantly higher at 34.0 ppt than at any other salinities. Plasma cortisol level was significantly higher but glucose level was lower at 1.7 ppt. Possessing TTX at a low salinity may be lethal to tiger puffer juveniles.

     

Simultaneous extraction and fractionation and thin layer chromatographic determination of 14 mycotoxins in grains.

A simple, systematic analytical method for multiple mycotoxins was developed for detecting 14 mycotoxins; aflatoxins B1, B2, G1, and G2, sterigmatocystin, T-2 toxin, diacetoxyscirpenol, neosolaniol, fusarenon X, zearalenone, ochratoxin A, citrinin, luteoskyrin, and rugulosin. These mycotoxins were extracted with 20% H2SO4-4% KCl-acetonitrile (2 + 20 + 178), defatted with isooctane, and transferred to chloroform. The chloroform extract was cleaned up by silica gel column chromatography; the first 10 toxins were eluted with chloroform-methanol (97 + 3) and the remaining 4 toxins with benzene-acetone-acetic acid (75 + 20 + 5). Each fraction was analyzed by thin layer chromatography for the final determination. The method has been applied to polished rice, rough rice, corn, wheat, and peanuts as an analytical screening procedure. The detection limits in these commodities ranged from 10.00 to 800.0 microgram/kg, depending on the mycotoxin, but all limits were superior to those obtained for the individual mycotoxins by using other methods.     

Effects of biotechnologically processed soybean meals in a nonfishmeal diet on growth performance, bile acid status, and morphological condition of the distal intestine and liver of rainbow trout Oncorhynchus mykiss

This study evaluated four commercially available biotechnologically processed soybean meals (SBMs) that were included as the primary protein sources in a nonfishmeal diet for juvenile rainbow trout Oncorhynchus mykiss. The processed SBMs were Afcep SBM fermented with mixed bacteria predominantly Bacillus spp., DaBomb SBM fermented with Lactobacillus acidophilus, PepSoyGen SBM fermented with Aspergillus oryzae and Bacillus subtilis, and HP300 SBM treated enzymatically. Fishmeal (FM) and common heat-treated and hulled SBM were used in the reference diets. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) analysis revealed that the protein components of Afcep SBM appeared to have relatively small molecular sizes compared with the other SBM products. After a 10-week feeding trial, growth of fish fed the common SBM diet was depressed, and abnormal liver and distal intestinal morphologies and bile acid characteristics were observed. The Afcep SBM diet showed approximated growth performance and comparable physiological conditions relative to the FM diet, while only limited improvements were observed in fish fed the other processed SBMs. These findings suggest that Afcep SBM, with its smaller molecular mass proteins, is the most promising processed SBM in nonfishmeal diets for rainbow trout.     

Changes in GnRH levels in the brain and pituitary gland during migrations of sockeye salmon and chum salmon

Levels of two moleculer types of gonadotropin-releasing hormone (GnRH), salmon GnRH (sGnRH) and chicken GnRH–II (cGnRH–II) in the various brain regions and pituitary gland of sockeye salmon (Oncorhynchus nerka) and chum salmon (O. keta) during smoltification and spawning migration, respectively, were measured using specific time-resolved fluoroimmunoassay (TR-FIA) systems. Changes in sGnRH levels in different brain regions tended to be specifically synchronized with serum thyroid hormone or pituitary gonadotropin (GTH) levels during smoltification and spawning migration, respectively. In contrast, cGnRH–II levels did not show such synchronized changes. SGnRH and cGnRH–II in various brain regions might have different roles during smoltification and spawning migration of salmonid fishes.     

Changes in the immunostaining intensities of follicle-stimulating hormone and luteinizing hormone during ovarian maturation in the female Japanese flounder

The role of gonadotropin (GTH) in the reproduction of the Japanese flounder, Paralichthys olivaceus, was studied by assessing the changes in the apparent activity of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in the pituitary gland during gonadal maturation by immunohistochemical analyses. Corresponding changes in plasma levels of testosterone (T), estradiol-17β (E₂), and 17α,20β-dihydroxy-4–pregnen-3-one (DHP) were also studied. Reared fish at the early spawning to termination stages were sampled from May to August and wild fish at the previtellogenic to termination stages were caught at 3- to 4-week intervals between April and September offshore from the northern mainland of Japan by gill nets. The gonadosomatic index of the reared fish decreased from the early spawning stage to the termination stage, while that of the wild fish increased significantly from the previtellogenic stage to the early spawning stage and decreased thereafter. In the reared fish, the immunostaining intensities of FSH and LH were high during the spawning period, accompanied by high plasma levels of T, E₂, and DHP. In the wild fish, the immunostaining intensities of FSH and LH were low during the previtellogenic stage but increased during the maturing and spawning stages. These results indicate that both FSH and LH are likely associated with oocyte maturation in the Japanese flounder.     

Isolation of an endophytic diazotroph,Klebsiella oxytoca,from sweet potato stems in Japan

Abstract

The population density of endophytic bacteria in the stem of field-grown sweet potato cultivars (Beniotome [BO], Koganesengan [KS], and Shiroyutaka [SYD in Miyakonojo, Miyazaki, Japan, ranged from 10² to 10⁴ cells g^?1 fresh weight sample using a semi-solid nitrogen-free medium. Eleven strains were isolated from the stems and two isolates, BO-1 and BO-5, showed a positive reaction in the acetylene reduction activity (ARA) test. BO-1 and BO-5 were isolated from cv. Beniotome in September 1999 and 2000, respectively. Morpho-physiological characterization of these isolates revealed that BO-1 and BO-5 showed a similar colony color in potato sucrose agar slants, produced bubbles in a modified semi-solid medium, acidified the medium, and displayed similar characteristics using the API 20NE rapid diagnostic kit. Partial sequence analysis of 16S rRNA from BO-1 revealed a 100% similarity (491 bp) to that of Klebsiella oxytoca. The other 9 isolates showed a negative reaction in the ARA test, slightly acidified or did not acidify the medium. Partial sequence analysis of the 16S rRNA revealed that the isolate SY-2 corresponded to Methylobacterium sp. (99.3% similarity for 1,241 bp), BO-3 to Pantoea agglomerans (99.1% similarity for 469 bp), and BO-8 to Sphingomonas sanguinis (98.8% similarity for 419 bp).     

Evidence for existence of cAMP-Epac signaling in the heads of mouse epididymal spermatozoa

Cyclic adenosine 3',5'-monophosphate (cAMP) signaling regulates the expression of fertilizing ability in mammalian spermatozoa. Many articles indicate that this signaling is mediated mainly via protein kinase A. Recently, a guanine nucleotide exchange factor for small G protein Rap1 (an exchange protein directly activated by cAMP: Epac) was discovered as a new mediator of cAMP signaling in somatic cells. The aim of this study was to reveal the existence of cAMP-Epac signaling in mouse spermatozoa. Northern blot analysis and in situ hybridization suggested that Epac1 and Epac2 mRNAs were transcribed in the seminiferous epithelia of the testis. This shows that expression of Epac mRNAs is present in mouse testicular germ cells. Indirect immunofluorescence with specific polyclonal antibodies suggested possible co-localization of Epac1 and Rap1 proteins in the heads of epididymal spermatozoa. Moreover, treatment of epididymal spermatozoa with an Epac-specific cAMP analog, 8-pMeOPT-2'-O-Me-cAMP, induced activation of Rap1, as revealed with a commercial kit for pull-down assay. These results indicate the existence of cAMP-Epac signaling in the heads of mouse epididymal spermatozoa.