Competitive enzyme-linked immunosorbent assay for the detection of the antibodies specific to akabane virus.

A competitive enzyme-linked immunosorbent assay (C-ELISA) using neutralizing monoclonal antibodies (MAbs) against Akabane virus (AKAV) was developed to detect antibodies to AKAV in cattle sera. The performance of the test using 7 different competitor MAbs was evaluated in sequential serum samples and sera from cattle infected with various bovine arboviruses. The dynamics of the antibody response expressed by percentage of inhibition (PI) in C-ELISA coincided with those of neutralizing antibody titers in sequential serum samples from 2 cattle experimentally infected with AKAV. The value of PI in C-ELISA for convalescent sera from cattle infected with arboviruses correlated with the neutralizing antibody titer to AKAV but was unaffected by the antibodies to other arboviruses. In the validation experiment of C-ELISA using 286 bovine sera previously examined for the AKAV antibody by serum neutralization (SN) test, the relative specificity of C-ELISA was more than 98%, whereas the relative sensitivities of individual MAbs ranged from 49% to 82.2%. Overall agreement between C-ELISA and the SN test varied from 72% to 90% depending on the MAb. These results suggest that the C-ELISA is acceptable as a rapid and specific method for detecting antibodies to AKAV and is a potential alternative to the SN test.     

Pathogenicity of emerging Japanese type 1 porcine reproductive and respiratory syndrome virus in experimentally infected pigs

To clarify the pathogenicity of Japanese type 1 porcine reproductive and respiratory syndrome virus (PRRSV) isolate in experimentally infected pigs, we evaluated clinical signs and monitored viremia for 21 days post-inoculation (dpi). Lungs were mottled, tanned and reddish in appearance; had lesions predominantly in the cranial, middle and accessory lobes; and failed to collapse at 10 dpi. Although microscopic lesions of lungs were reproduced using the Japanese emerging type 1 PRRSV isolate under experimental conditions, no significant differences were noted between the challenge and control groups regarding mean rectal temperature and daily weight gain. These results provide useful insights into the limited pathogenicity of single infection with the Japanese type 1 PRRSV isolate in piglets, which differ from findings in reported field cases.     

Characterization of the quantitatively major collagen in the mantle of oyster <Emphasis Type="Italic">Crassostrea gigas</Emphasis>

ABSTRACT:   A quantitatively major collagen was isolated from the pepsin-solubilized collagen preparation of the mantle by differential salt precipitation and phosphocellulose column chromatography, and its constituent α components (α1 and α2) were purified by phosphocellulose column chromatography. The subunits were demonstrated to be genetically distinct from each other by peptide mapping and amino acid analysis. The amino acid composition calculated from those of the α1 and α2 components in 2 : 1 ratio coincided well with that of the major collagen from the mantle. These results suggest that the major collagen in the mantle of the oyster may have a heterotrimer structure (α1)₂α2.     

Dynamic excitation and static loading tests of glulam lattice floor

A wooden lattice floor with high stiffness and damping capacity has been developed to solve noise problems in wooden apartment houses. The lattice floor consisted of Douglas fir glulam beams with inserted steel plate joints and drift pins. To examine the structural performance of the floor, dynamic excitation and static loading tests were conducted on the full size floor. The first and second order resonance frequencies of the floor were 13.5Hz and 27.0Hz, respectively. These frequencies are similar to the peak frequency of a conventional wooden floor and the combined floor fabricated from glued laminated timber and iron. The maximum static load of the floor was 127kN. The apparent flexural rigidity was less than half the value of several floors studied in the past. However, it is considered that the stiffness is improved by constructing panels and this floor has almost equivalent performance. Relative deflection was not affected by the loading history.Part of this study was presented at the International Wood Engineering Conference, New Orleans, October 1996.     

Census and effective population sizes of white‐spotted charr (Salvelinus leucomaenis) in a fragmented landscape

Several habitat models have been proposed to predict population size for stream fishes and to guide habitat assessment and monitoring techniques. However, most models do not incorporate the potential advantage of molecular genetic markers. We conducted a field survey and microsatellite DNA analyses to quantify the relationships among genetic diversity, census/effective population size and habitat variables in fragmented populations of white‐spotted charr (Salvelinus leucomaenis). The census population size significantly increased with the stream length, the number of pools and a pool‐riffle sequence index, a proxy for channel‐unit habitat type complexity within reaches. Population density was correlated with the pool‐riffle sequence index only. Genetic diversity and effective population size were not correlated with the habitat variables or census population size. There was a lack of isolation‐by‐distance population structure in the studied populations. Our results suggest that stream length and the number of pools within reaches associated with habitat complexity are the habitat variables that explain the majority of variation in population size of white‐spotted charr. Our findings provide further evidence that census population size per se is a poor indicator of the inclusive genetic diversity within populations in a fragmented landscape.     

Hemorrhagic necrotizing splenitis in a slaughter pig infected with Arcanobacterium species

A 6-month-old barrow presented with lethargy, inappetence and dysstasia. At necropsy, multiple coalescing hemorrhagic foci were detected in the margins of the spleen. Gram-positive bacilli were isolated from the spleen, kidney, muscle and liver. Comparative 16S rDNA gene sequencing analysis of the isolates (TO16177) revealed that they would be the same species of unpublished Arcanobacterium species strain HJ57-14E (accession no. gi 18873551) (99.7% similarity based on a comparison of 675 bp). Histologic examination of the splenic tissue sections revealed extensive necrosis and inflammation, and gram-positive bacilli were discernible. Multifocal necrosis was also detected in the liver. Immunohistochemically, the isolates were cross-reacted with polyclonal antibodies against Arcanobacterium pyogenes and Actinomyces naeslundii, and the reaction was strong for the latter. Similar reactions were found in the suppurative lesions of the tonsil, and occasionally in the spleen and lymph nodes. The present results indicate that the unpublished Arcanobacterium species induced multiple organ failure accompanied by acute hemorrhagic necrotizing splenitis in this growing-finishing pig.     

Spindle formation and microtubule organization during first division in reconstructed rat embryos produced by somatic cell nuclear transfer

The present study was conducted to demonstrate the spindle formation and behavior of chromosomes and microtubules during first division in reconstructed rat embryos produced by somatic cell nuclear transfer (SCNT) with cumulus cell nuclei. To demonstrate the effect of oocyte aging after ovulation on the cleavage of SCNT embryos, micromanipulation was carried out 11, 15 and 18 h after injection of hCG. SCNT oocytes were activated by incubation in culture medium supplemented with 5 microM ionomycin for 5 min followed by treatment with 2 mM 6-dimethylaminopurine (6-DMAP) in mR1ECM for 2-3 h. For immunocytochemical observation, the SCNT embryos were incubated with monoclonal anti-alpha-tubulin antibody and then fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG. Cleavage rates were significantly higher for oocytes collected after 15 and 18 h rather than for those collected 11 h after injection of hCG (56 and 53%, respectively vs. 28%; P<0.05). Premature chromosome condensation occurred before activation of the SCNT oocytes, but adequate spindle formation was only rarely observed. The distribution of microtubules in SCNT embryos after activation was different from those of fertilized and parthenogenic oocytes, i.e., a dense microtubule organization shaped like a ring was observed. Eighteen to 20 h post-activation, most SCNT embryos were in the 2-cell stage, but no nucleoli were clearly visible, which was quite different from the fertilized oocytes. In addition, first division with and without small cellular bodies containing DNA was observed in the rat SCNT embryos in some cases. The present study suggests that reorganization of transferred nuclei in rat SCNT embryos may be inadequate in terms of formation of the mitotic assembly and nucleolar reorganization.     

Comparison of the characters of the plaque-purified viruses from foot-and-mouth disease virus O/JPN/2000

At least two biotypes were observed at the 2nd passage stage after the isolation of Foot-and-mouth disease Virus (FMDV) O/JPN/2000 strain. These 2 types of viruses differed from their plaque phenotypes and were distinguishable by using a monoclonal antibody (MAb) 64G8 that was made for the FMDV O/JPN/2000 strain. One of these 2 biotypes formed small plaque (SP) and with immuno staining showed a positive reaction to MAb 64G8, while the other formed clear large plaque (LP) and did not react with MAb 64G8. The amino acid sequences of the capsid coding region (VP1-VP4) of the SP virus (SPV) and the LP virus (LPV) revealed two substitutions on the 133rd amino acid in VP2, and the 56th amino acid in VP3. These amino acid changes of SPV and LPV are Asn to Asp, Arg to His, respectively. The Arg of the 56th amino acid in VP3 that have been known as critical position of cell culture adapted virus. Only LPV showed high pathogenicity in suckling mice, and its LD(50) was calculated to be about 10(2) TCID(50)/0.1 ml. These results showed that the SPV that existed at the 2nd passage stage from isolation was a low virulence virus, which may suggest why the pathogenicity of O/JPN/2000 did not show clear symptoms in infected cattle.     

猪隐秘杆菌型出血性坏死性脾炎

六月龄去势公猪表现昏睡、食欲不振、站立困难。尸检发现脾脏边缘呈多重出血灶。从脾脏、肾脏、肌肉和肝脏中分离出革兰氏阳性杆菌,对分离株(TO16177)的16S rDNA基因序列比较分析发现,其可能与未发表过的隐秘杆菌属HJ57-14E菌株(登记号:gi18873551)(比较675bp个碱基,相似性达99.7%)为同一个属。脾脏组织切片的组织学检查发现呈广泛性坏死和炎症,其中革兰氏阳性杆菌显而易见。肝脏中可见多病灶的坏死斑。免疫组织化学检测发现,分离株与抗化脓性隐秘杆菌属(Arcanobacterium pyogenes)和内氏放线菌(Actinomyces naeslundii)的多克隆抗体具有交叉反应,且与后者的交叉反应更强烈。相似的反应也见于扁桃体的化脓灶中分离株,偶尔也见于脾脏和淋巴结中的分离株。本研究结果表明,这种未公开发布的隐秘杆菌属的细菌会引起生长肥育猪多器官功能衰竭,而后继发急性出血性坏死性脾炎。     

Caprine somatic cell nuclear transfer using in vivo matured oocytes collected by laparoscopic follicular aspiration

In vivo matured oocytes collected by laparoscopic follicular aspiration (LFA) from hormone treated female goats were used as recipient ooplasts for somatic cell nuclear transfer (SCNT). Japanese native (Shiba) goats were used as donor females and some donor females were used repeatedly (two or three times) at intervals of a few months. To induce synchronization of estrus, a sponge containing 0.5 g of progesterone was inserted into the vagina of each goat for 14 days. These animals were also treated with follicle stimulating hormone (FSH) in a series of 8 injections over 4 days. The first FSH injection was administered on the morning of day 9 of sponge insertion. On the morning of day 13, 50 µg of gonadotropin‐releasing hormone (GnRH) was injected into each animal. Twenty‐nine hours after GnRH injection, LFA was performed. After removal of cumulus cells, collected oocytes with the first polar body were selected and enucleated for nuclear transfer. Anterior pituitary cells isolated from an adult male Shiba goat were transfected with a DNA fragment containing the enhanced green flourescent protein gene and the puromycin resistance gene. A single donor cell was inserted into the perivitelline space of each enucleated oocyte and fusion was induced with one electric pulse of 20 V for 10 µs. The SCNT goat eggs were cultured in chemically defined medium at 38.5°C in 5% CO₂, 5% O₂, 90% N₂ for 9 days. By LFA, 396 oocytes were collected from a total of 30 females. After removal of cumulus cells, 64% of them extruded the first polar body. The percentage of SCNT goat eggs produced using in vivo matured oocytes which developed to the blastocyst stage (20–21%) was significantly higher (P < 0.05) than that produced with in vitro matured oocytes (3–8%).