Chicken peripheral blood CD3+CD4-CD8- cells are regulated by endocrine and nerve systems

The existence of CD3(+)CD4(-)CD8(-) T cells in thymus and spleen has already been known. However, because of the presence of large amounts of thrombocytes in peripheral blood (PB), the proportion of CD3(+)CD4(-)CD8(-) T cells in PB has yet to be investigated. Therefore, the proportion of peripheral T cell-subsets was investigated in 6-week-old chickens. The percentage of CD3(+) cells, CD4(+) cells, CD8 alpha(+) cells, CD8 beta(+), and CD3(+)CD4(-)CD8(-) cells was 76%, 41%, 14%, 5%, and 15%, respectively. The proportion of CD3(+)CD4(-)CD8(-) cells in PB increased during egg-laying periods and in chickens treated with an analog of estrogen, while it decreased with age and in response to restraint stress. All of the CD3(+)CD4(-)CD8(-) cells expressed TCR1, and did not have NK activity. CD3(+)CD4(-)CD8(-) cells represent about 60% of peripheral TCR1(+) cells. These findings indicate that the proportion of CD3(+)CD4(-)CD8(-) cells is regulated by the endocrine and nerve systems.     

Developmental competence of porcine blastocysts produced in vitro

The establishment of in vitro embryo production (IVP) system in pigs enables us to generate viable embryos with a quality equal to that of in vivo derived embryos. This technology contributes not only to developments in reproductive physiology and agriculture but also to biotechnologies for producing cloned or genetically modified pigs. The birth of piglets from in vitro matured and fertilized embryos at the two- to 4-cell stage was first achieved about 10 years ago, but it was only quite recently that piglets were produced after the transfer of IVP blastocysts. This improvement to the blastocyst stage of the in vitro culture system after in vitro maturation and fertilization can be expected to play a part in the development of an advanced IVP system. Here, we discuss the developmental ability of porcine embryos produced by our improved IVP system and the utilization of this technique in the new biotechnology.     

Seroprevalence of Bartonella infection in American free-ranging and captive pumas (Felis concolor) and bobcats (Lynx rufus)

Bartonella henselae is the main agent of cat scratch disease in humans and domestic cats are the main reservoir of this bacterium. We conducted a serosurvey to investigate the role of American wild felids as a potential reservoir of Bartonella species. A total of 479 samples (439 serum samples and 40 Nobuto strips) collected between 1984 and 1999 from pumas (Felis concolor) and 91 samples (58 serum samples and 33 Nobuto strips) collected from bobcats (Lynx rufus) in North America, Central America and South America were screened for B. henselae antibodies. The overall prevalence of B. henselae antibodies was respectively 19.4% in pumas and 23.1% in bobcats, with regional variations. In the USA, pumas from the southwestern states were more likely to be seropositive for B. henselae (prevalence ratio (PR) = 2.82, 95% confidence interval (CI) = 1.55, 5.11) than pumas from the Northwest and Mountain states. Similarly, adults were more likely to be B. henselae seropositive than juveniles and kittens (PR = 1.77, 95% CI = 1.07, 2.93). Adult pumas were more likely to have higher B. henselae antibody titers than juveniles and kittens (p = 0.026). B. henselae antibody prevalence was 22.4% (19/85) in bobcats from the USA and 33.3% (2/6) in the Mexican bobcats. In the USA, antibody prevalence varied depending on the geographical origin of the bobcats. In California, the highest prevalence was in bobcats from the coastal range (37.5%). These results suggest a potential role of wild felids in the epidemiological cycle of Bartonella henselae or closely related Bartonella species.     

In vitro and in vivo developmental ability of oocytes derived from porcine primordial follicles xenografted into nude mice

We evaluated the developmental ability of oocytes in porcine primordial follicles xenografted into nude mice. Ovarian tissues from 20-day-old piglets, in which most of the follicles were primordial, were transplanted under the kidney capsules of ovariectomized nude mice. Forty-nine to 89 days after grafting (mean +/- SEM, 66.9 +/- 1.9 days; n = 64), the host mice showed the presence of cornified epithelial cells in their vaginal smears for the first time. The mice were then treated with 4 IU of equine chorionic gonadotropin (eCG) 60 days after first detection of vaginal cornification. Oocytes were collected from the host mice 48 h after treatment with eCG, and then matured. The maturation rates, based on the incidence of first polar body, ranged from 25.1% to 42.5%. They were then fertilized in vitro and cultured in vitro for 6 days, or transferred into estrous-synchronized recipients and recovered after 6 days. On Day 6 of culture, 15.4% of the matured oocytes had cleaved to the 2- to 8-cell stage. However, neither the embryos cultured in vitro nor those transferred and recovered developed to advanced embryonic stages, such as morulae or blastocysts. This result suggests that the developmental ability of xenografted oocytes is insufficient, even after in vitro maturation. Further strategies, such as improvement of hormonal treatment for host mice, are required to enable oocytes in xenografted ovarian tissues to acquire the cytoplasmic maturation necessary for embryonic development.     

Design of hammerhead ribozymes that cleave murine Sry mRNA in vitro and in vivo

As the first step in investigating the possiblity of applying ribozyme technology to artificial control of the sex ratios at birth in farm animals, where the demand for females exceeds that for males, we designed a hammerhead ribozyme (HHRz) and 2 tRNA(val)-hammerhead ribozyme complexes (tRNARz3 and tRNARz4), and examined their effects upon murine Sry mRNA in vitro and in cells. We demonstrated that HHRz and tRNARz3 could effectively cleave the target Sry mRNA in vitro. For the purpose of experiments in vivo, HHRz was cloned into the highly efficient pUC-CAGGS mammalian expression vector (pCAG/HHRz), and the tRNA ribozyme complexes were cloned into the pol III promoter-driven pPUR-KE vector (pPUR/tRNARz3 and pPUR/tRNARz4); the ribozyme vectors were co-transfected with the target vector (pCAG/Sry). A suppressive action (up to approx. 60%) was confirmed for pCAG/HHRz and pPUR/tRNARz3 upon the transiently expressed exogenously introduced Sry in M15 cultured cells.     

Characterization of a new Maillard type reaction product generated by heating 1-deoxymaltulosyl-glycine in the presence of cysteine

The reaction between Amadori compounds and cysteine was investigated. When 1-deoxymaltulosyl-glycine (glycyl-fructosyl-glucose) was heated at 100 degrees C with cysteine in a neutral aqueous solution, a novel intermediate composed of 1-deoxyosone and cysteine was detected. NMR and mass spectrometry studies revealed the structure of the isolated intermediate to be 7,8a-dihydroxy-4a-methyl-8-(alpha-d-glucopyranosyloxy)hexahydro-5-oxa-4-thia-1-azanaphthalene-2-carboxylic acid. This intermediate easily generated isomaltol and acetylfuran as volatile compounds in 1 mol/L HCl at 100 degrees C.     

A newly established bovine intestinal epithelial cell line is effective for in vitro screening of potential antiviral immunobiotic microorganisms for cattle

We evaluated whether a bovine intestinal epithelial (BIE) cell line could serve as a useful in vitro model system for studying antiviral immune responses in bovine intestinal epithelial cells (IECs) and for the primary screening of immunobiotic microorganisms with antiviral protective capabilities. Immunofluorescent analyses revealed that toll-like receptor 3 (TLR3) was expressed in BIE cells, and the results of real-time quantitative PCR showed that these cells respond to stimulation with poly(I:C) by up-regulating pro-inflammatory cytokines and type I interferons. In addition, we demonstrated that BIE cells are useful for the primary screening of immunobiotic lactic acid bacteria strains which are able to beneficially modulate antiviral immune responses triggered by TLR3 activation in bovine IECs. The characterization of BIE cells performed in the present study represents an important step towards the establishment of a valuable bovine in vitro system that could be used for the development of immunomodulatory feed for bovine hosts.     

Effects of five oleanolic acid triterpenoid saponins from the rhizome of Anemone raddeana on stimulus-induced superoxide generation, phosphorylation of proteins and translocation of cytosolic compounds to cell membrane in human neutrophils

Five oleanolic acid triterpenoid saponins (OTS-1, 2, 3, 4 and 5) were isolated from the rhizome of Anemone raddeana. The effect of these triterpenoid saponins on stimulus-induced superoxide generation in human neutrophils was assayed by measuring the reduction of ferricytochrome c using a dual-beam spectrophotometer. The phosphorylation of neutrophil proteins, and translocation of p67(phox), p47(phox) and Rac to plasma membrane were investigated using specific monoclonal antibodies. The five oleanolic acid triterpenoid saponins used in this experiment suppressed N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced superoxide generation in a concentration-dependent manner. OTS-1, 2 and 4 suppressed phorbol 12-myristate 13-acetate (PMA)- and arachidonic acid (AA)-induced superoxide generation in a concentration-dependent manner, but OTS-3 and 5 showed no effect. fMLP- and PMA-induced tyrosyl or serine/threonine phosphorylation, and fMLP-, PMA- and AA-induced translocation of p67(phox), p47(phox) and Rac to plasma membrane were in parallel with the suppression of the stimulus-induced superoxide generation.     

Vertical Transmission of Brucella abortus Causes Sterility in Pregnant Mice

The mechanisms of abortion and sterility induced by bacterial infection are largely unknown. In the present study, we found that Brucella abortus, a causative agent of brucellosis and facultative intracellular pathogen, caused sterility in pregnant mice. We have recently established a mouse model for abortion induced by B. abortus infection and high rates of abortion are observed for bacterial infection on day 4.5 of gestation, but not for other days. Infected newborn (first generation) mice showed poor growth compared with uninfected newborn mice and bacterial replication in the spleen of the former was observed over a long period. When infected first generation female mice were mated to infected first generation male mice, the number of fetuses was significantly less than that in uninfected first generation mice. These infected second generation mice also showed poor growth. These results suggest that vertical transmission of B. abostus causes sterility in pregnant mice and our mouse model would be useful for the investigating of brucellosis.     

Efficacy of a single intravenous dose of the neuraminidase inhibitor peramivir in the treatment of equine influenza

Equine influenza A virus (EIV) of the H3N8 subtype is an important pathogen causing acute respiratory disease in horses. Peramivir is a selective inhibitor of the influenza virus neuraminidase (NA). The characteristics of peramivir are not only its capacity for parenteral administration, but also its strong affinity for NA and slow off-rate from the NA-peramivir complex, suggesting that it could lead to a prolonged inhibitory effect and thus allow a lower dosing frequency. The aims of this study were to evaluate the inhibitory efficacy of peramivir against the NA activities of EIV in vitro and the treatment efficacy of a single intravenous dose of peramivir in horses experimentally infected with EIV. Peramivir inhibited the activities of NA from the seven contemporary EIV strains in vitro, with 50% inhibitory concentrations ranging from 0.10 to 0.20nmol/L. Horses treated with a single IV dose of peramivir (3000mg/600mL/animal, 7.8-9.3mg/kg of bodyweight) showed significantly milder clinical signs (pyrexia, nasal discharge and cough) with a shorter duration than control horses injected with normal saline. Moreover, the mean duration of virus shedding for the horses treated with peramivir was significantly shorter than for the control horses. These findings suggested that a single IV administration of peramivir had good potential for the treatment of equine influenza, and may help to limit the spread of the disease in the horse population.