Development of an enzyme-linked immunosorbent assay for the measurement of antibodies against infectious coryza vaccine

Infectious coryza is an acute respiratory disease caused by infection with Avibacterium (Haemophilus) paragallinarum. It is characterized by nasal discharge and facial swelling and is associated with growth retardation and a reduction in egg production. Hemagglutination inhibition (HI) tests are used to estimate vaccine-induced immunity against infectious coryza in vitro; however, these procedures are complicated and their sensitivity is insufficient. To address these problems, an enzyme-linked immunosorbent assay (ELISA) technique using serovar-specific regions of HMTp210 (210 kDa), an outer-membrane protein of A. paragallinarum, was developed to measure the antibodies against infectious coryza. Chickens with an ELISA titer of 0.3 or more did not exhibit clinical signs of infectious coryza against challenge with A. paragallinarum, although their HI antibody titers were negative. On the other hand, chickens with an ELISA titer below 0.3 exhibited clinical signs of the disease with one exception. Antibody prevalence rates on ELISA were 80% and 60% against infection with serovars A and C, respectively, and ELISA also detected antibodies in chickens infected with A. paragallinarum with a sensitivity higher than that of HI tests. Taken together, the ELISA technique developed in this study is a valuable tool for the measurement of antibodies produced against the infectious coryza vaccine or in response to an infection with A. paragallinarum.     

Evaluation of Al-tolerance on upland and lowland types of NERICA lines under hydroponic conditions

We evaluated Al-tolerance in 44 interspecific lines (32 upland and 12 lowland) developed from the crosses of Oryza sativa and O. glaberrima called New Rice for Africa (NERICA) with 2 O. glaberrima lines and 13 O. sativa varieties under hydroponic culture containing 0.15, 0.3, 0.6, and 1.2 mM Al (+Al) and 0 mM Al (?Al as a control). Ten upland and four lowland NERICA lines showed strong Al-tolerance judging from their higher relative root and shoot dry weights (percentage ratios of dry weights in the Al treatments to the control) than those of the tolerant O. sativa check of IR 53650. Their tolerance was supported by relatively higher root Al accumulation (dark blue color) opposite performance with common knowledge (shown pale blue color) in root using hematoxylin staining compared to the Al-susceptible genotypes identified based on relative root and shoot dry weights in the study. Net Al concentration was higher in roots than in shoots in all +Al conditions for all genotypes; however, a clear difference in the Al concentration among the Al-tolerant, Al-moderately tolerant, and Al-susceptible genotypes was observed in the shoots. Al concentrations in the shoots of the Al-tolerant and Al-moderately tolerant upland and lowland NERICA lines were significantly lower than those of its Al-susceptible counterparts in the groups under 0.6 and 1.2 mM Al conditions, respectively. Differences in root and shoot growth among the Al-tolerant, Al-moderately tolerant, and Al-susceptible NERICA lines were clearer under strong Al toxic conditions (0.6 and 1.2 mM Al) than under weak Al toxic conditions (0.15 and 0.3 mM Al).     

Prevalence of canine distemper virus, feline immunodeficiency virus and feline leukemia virus in captive African lions (Panthera leo) in Japan

Sero-prevalences of canine distemper virus (CDV), feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) were evaluated in 20 captive lions in two Japanese zoos. Anti-CDV antibody was detected in 13 of 20 lions. We could pursue antibody responses against CDV in three lions back to 1996. Sera collected in 1996 were negative for anti-CDV antibody, therefore, all of them showed sero-conversion in 2000. This result suggested that the epidemic of CDV infection in this zoo might have happened between 1996 and 2000. The lions were also examined for FIV and FeLV infections. We had no evidence for FeLV infection but eight lions were sero-positive for anti-FIV antibody.     

Passage of equine herpesvirus-1 in suckling mouse brain enhances extraneural virus growth and subsequent hematogenous neuroinvasion

Intracerebral inoculation of field-isolates as well as established strains of equine herpesvirus-1 (EHV-1) in suckling mice results in viral replication in neurons and glial cells and induces encephalitis. By intraperitoneal (i.p.) inoculation, no histological lesion was observed in the central nervous system (CNS) in suckling mice with the EHV-1 HH1 strain (HH1), whereas a neuroadapted variant (NHH1) produced by serial passage of HH1 in the mouse brain caused severe encephalomyelitis after i.p. inoculation. The purpose of this study was to determine the route of neuroinvasion after i.p. inoculation of NHH1 and to clarify the effects of the brain passage on viral neuroinvasion. NHH1, but not HH1, targeted splenic and pulmonary macrophages and omental fat cells on days 1 and 2 post-inoculation (p.i.). From days 1 to 3 p.i., cell-associated viremia was occurred in NHH1-infected mice, but not in HH1-infected mice. On day 4 p.i., viral antigen was detected in a few endothelial cells, perivascular glial cells and neurons in the CNS in NHH1-infected mice. The number of viral antigen-positive cells increased markedly after day 5 p.i. In contrast, no viral antigen-positive cell was detected in the CNS in HH1-infected mice, except for a few nerve cells in the thoracic cord on day 4 p.i. These results suggest that NHH1 neuroinvasion is hematogenous and is correlated with enhanced extraneural virus growth.     

Increase in muscle endurance in mice by dietary Yamabushitake mushroom (Hericium erinaceus) possibly via activation of PPARδ

Skeletal muscle fiber is largely classified into two types: type 1 (slow‐twitch) and type 2 (fast‐twitch) fibers. Meat quality and composition of fiber types are thought to be closely related. Previous research showed that overexpression of constitutively active peroxisome proliferator‐activated receptor (PPAR)δ, a nuclear receptor present in skeletal muscle, increased type 1 fibers in mice. In this study, we found that hexane extracts of Yamabushitake mushroom (Hericium erinaceus) showed PPARδ agonistic activity in vitro. Eight‐week‐old C57BL/6J mice were fed a diet supplemented with 5% (w/w) freeze‐dried Yamabushitake mushroom for 24 hr. After the treatment period, the extensor digitorum longus (EDL) muscles were excised. The Yamabushitake‐supplemented diet up‐regulated the PPARδ target genes Pdk4 and Ucp3 in mouse skeletal muscles in vivo. Furthermore, feeding the Yamabushitake‐supplemented diet to mice for 8 weeks resulted in a significant increase in muscle endurance. These results indicate that Yamabushitake mushroom contains PPARδ agonistic ligands and that dietary intake of Yamabushitake mushroom could activate PPARδ in skeletal muscle of mice. Unexpectedly, we observed no significant alterations in composition of muscle fiber types between the mice fed control and Yamabushitake‐supplemented diets.     

Cell proliferation potency is independent of FGF4 signaling in trophoblast stem cells derived from androgenetic embryos

We previously established trophoblast stem cells from mouse androgenetic embryos (AGTS cells). In this study, to further characterize AGTS cells, we compared cell proliferation activity between trophoblast stem (TS) cells and AGTS cells under fibroblast growth factor 4 (FGF4) signaling. TS cells continued to proliferate and maintained mitotic cell division in the presence of FGF4. After FGF4 deprivation, the cell proliferation stopped, the rate of M-phase cells decreased, and trophoblast giant cells formed. In contrast, some of AGTS cells continued to proliferate, and the rate of M-phase cells did not decrease after FGF4 deprivation, although the other cells differentiated into giant cells. RO3306, an ATP competitor that selectively inhibits CDK1, inhibited the cell proliferation of both TS and AGTS cells. Under RO3306 treatment, cell death was induced in AGTS cells but not in TS cells. These results indicate that RO3306 caused TS cells to shift mitotic cell division to endoreduplication but that some of AGTS cells did not shift to endoreduplication and induced cell death. In conclusion, the paternal genome facilitated the proliferation of trophoblast cells without FGF4 signaling.     

The growth‐promoting activity of egg white proteins in the C2C12 myoblast cell line

In this study, we examined the effects of several egg white proteins (ovalbumin, ovomucoid, ovotransferrin and lysozyme) on proliferation and myotube growth in C2C12 murine myoblast cells. Cell proliferation was measured using a water‐soluble tetrazolium salt (WST‐8)‐based assay and then validated using Giemsa staining. Significant proliferative activities of C2C12 cells were observed in response to the addition of 10^?5–10^?4 mol/L ovalbumin or ovomucoid. Ovotransferrin decreased C2C12 cell proliferation and lysozyme showed no significant effects on the proliferation of C2C12 cells. In contrast, the proliferative effects of ovalbumin and ovomucoid were not observed in 3T3‐L1 murine preadipocyte cells. We also measured the effects of ovalbumin and ovomucoid on C2C12 myotube diameters by using histological analysis. In comparison to control cells, myotube diameters were significantly increased in cells cultured in 10^?6–10^?4 mol/L ovalbumin or ovomucoid, suggesting that ovalbumin and ovomucoid stimulate the growth of myotubes. Thus, our results clearly demonstrated that ovalbumin or ovomucoid stimulated the proliferation of myoblasts and growth of myotubes.     

Mechano-biology of skeletal muscle hypertrophy and regeneration: Possible mechanism of stretch-induced activation of resident myogenic stem cells

In undamaged postnatal muscle fibers with normal contraction and relaxation activities, quiescent satellite cells of resident myogenic stem cells are interposed between the overlying external lamina and the sarcolemma of a subjacent mature muscle fiber. When muscle is injured, exercised, overused or mechanically stretched, these cells are activated to enter the cell proliferation cycle, divide, differentiate, and fuse with the adjacent muscle fiber, and are responsible for regeneration and work-induced hypertrophy of muscle fibers. Therefore, a mechanism must exist to translate mechanical changes in muscle tissue into chemical signals that can activate satellite cells. Recent studies of satellite cells or single muscle fibers in culture and in vivo demonstrated the essential role of hepatocyte growth factor (HGF) and nitric oxide (NO) radical in the activation pathway. These experiments have also reported that mechanically stretching satellite cells or living skeletal muscles triggers the activation by rapid release of HGF from its extracellular tethering and the subsequent presentation to the receptor c-met. HGF release has been shown to rely on calcium-calmodulin formation and NO radical production in satellite cells and/or muscle fibers in response to the mechanical perturbation, and depend on the subsequent up-regulation of matrix metalloproteinase (MMP) activity. These results indicate that the activation mechanism is a cascade of events including calcium ion influx, calcium-calmodulin formation, NO synthase activation, NO radical production, MMP activation, HGF release and binding to c-met. Better understanding of 'mechano-biology' on the satellite cell activation is essential for designing procedures that could enhance muscle growth and repair activities in meat-animal agriculture and also in neuromuscular disease and aging in humans.     

In vitro measurement of post‐natal changes in proliferating satellite cell frequency during rat muscle growth

Satellite cells, resident myogenic stem cells found in postnatal skeletal muscle, are most abundant during early postnatal development and sharply decline in frequency thereafter to adult levels in mice and rats. Therefore, postnatal changes in satellite cell mitotic activities are important aspects for further understanding a muscle growth strategy. In large meat‐production animals, however, the traditional in vivo proliferation assay may be less realistic because it requires intra‐peritoneal (ip) injection of huge dosage of mutagenic nucleosides, ³H‐labeled thymidine or bromodeoxyuridine (BrdU), at each age‐time of sacrifice. We report in the present pilot study using rats that in vivo proliferation activity of satellite cells can be evaluated by an in vitro BrdU‐incorporation assay in early cultures. Briefly, satellite cells were prepared from upper hind‐limb and back muscles and maintained for 24 h with imposing by BrdU addition for the last 2 h, followed by the regular immunocytochemistry for determining BrdU‐incorporated cell percentage. This in vitro assay demonstrated a rapid decrease in proliferating satellite cell frequency to the adult level during about 3‐month period after birth, and yielded a high correlation to the measurements by the in vivo BrdU ip‐injection method during the postnatal period examined from day‐2 to month‐11. The in vitro proliferation assay may be further adaptable for large domestic animals by the combination with a muscle biopsy technique that enables age‐interval sampling from the same growing animals.     

Calf production from vitrified bovine sexed embryos following in‐straw dilution

The objective of this study was to develop an in‐straw dilution method suitable for direct transfer of vitrified bovine sexed embryos. Embryo sexing was performed by molecular diagnosis. Several sexed and vitrified‐warmed embryos were transferred after evaluation of morphologically embryonic survival at warming and in‐straw dilution (Evaluation group). The other embryos were immediately directly transferred to recipients without first being expelled from the straws after in‐straw dilution (Non‐evaluation group). The pregnancy rates of vitrified sexed embryos were 38.7% and 34.8% in the Evaluation group and Non‐evaluation group, respectively, which were not significantly different. The viability of lower quality embryos before vitrification tended to be lower (P = 0.087) than that of the higher quality embryos regardless of evaluating embryos after warming and in‐straw dilution. The abortion rates were similar, and there was no difference between the two groups (13.9% and 12.5%, respectively). These results demonstrate that vitrified bovine sexed embryos can be vitrified and diluted by the in‐straw method and that the vitrified and warmed sexed embryos can develop to term.