Antitumor effect of adriamycin entrapped in liposomes conjugated with anti-bovine tumor antigen monoclonal antibody in leukemic cows

Monoclonal antibody c143 against tumor-associated antigen (TAA) expressed on bovine leukemia cells was conjugated to liposomes containing adriamycin (ADM), and therapeutic effects of conjugates were examined in leukemic or preleukemic cows to prevent the progression of the disease. Five cows with TAA-positive in their peripheral blood lymphocytes were divided into two groups. Each group of cows received 4 injections of ADM alone (0.4 mg/kg) or c143-conjugated liposomes containing the same dose of ADM (L-AMD-c143) through the jugular vein at about 4 day intervals. In three animals treated with L-ADM-c143, the TAA-positive cells gradually decreased with treatment and finally two animals became TAA-negative during a 6 week period and a 14 week period after treatment, respectively. In the control, two animals treated with ADM alone showed only a slight decrease of TAA-positive cells.     

Establishment of a quantitative ELISA for the measurement of allergen-specific IgE in dogs using anti-IgE antibody cross-reactive to mouse and dog IgE

As IgE plays a pivotal role in type I hypersensitivity-mediated allergic diseases, it is valuable to measure absolute quantity of serum antigen-specific IgE for clinical and research purposes. Here we describe a novel ELISA system that enables quantification of antigen-specific IgE in ng/ml in dogs. A newly developed monoclonal antibody (CRE-DM) was shown to recognize canine and mouse IgE equally in a dose dependent manner, but it did not recognize canine IgG. The reactivity of CRE-DM to canine IgE was also confirmed by an inhibition ELISA using canine IgE as an inhibitor and the maximum inhibition rate was 91.3%. In order to know whether canine IgE specific to an allergen could be quantitatively measured with an ELISA using CRE-DM, we established a quantitative ELISA that could measure canine IgE recognizing Cry j 1, one of the major allergens of Japanese cedar pollen. In this ELISA, a standard curve was created by using concentration-predetermined Cry j 1-specific monoclonal mouse IgE. According to the standard curve, the concentration of Cry j 1-specific IgE in dogs that were experimentally sensitized to Japanese cedar pollen could be calculated and determined in ng/ml. The specificity of the Cry j 1-specific IgE ELISA using CRE-DM was also confirmed by inhibition ELISA using canine IgE as an inhibitor and the inhibition rate was 97.0%. Reproducibility of the ELISA in three independent assays was determined using groups of pooled canine sera whose Cry j 1-IgE titers ranged from 155.9 to 888.2 ng/ml. Intra- and inter-assay reproducibility was determined with coefficient of variation ranging between 3.1-5.2% and 2.2-8.0%, respectively. These results demonstrated that the ELISA utilizing CRE-DM was a specific, reliable and robust new laboratory test that could quantify absolute amount of antigen-specific IgE in canine serum. The ELISA will serve as a useful tool in the clinics to evaluate the change of serum IgE titers during anti-allergic treatments as well as during seasonal fluctuation of allergen exposure.     

Optimization of conditions for in vitro production of radical oxygen species and expression of tissue factor by canine mononuclear cells and granulocytes for use in high-throughput assays

The purpose of this study was to optimize conditions for high throughput measurement of radical oxygen species (ROS) production and expression of tissue factor, also termed procoagulant activity, by canine leukocytes. Granulocytes and mononuclear cells were separated by density gradient centrifugation from peripheral blood collected on several occasions from three healthy large breed dogs. To determine optimal conditions for ROS production, granulocytes were incubated for 1 or 3h in PBG (PBS containing 0.5% BSA and 5mM glucose) or RPMI containing 10% fetal bovine serum (FBS); lipopolysaccharide (LPS), zymosan, peptidoglycan (PGN) and phorbol myristate acetate (PMA) were used as stimuli. ROS was assessed by conversion of the nonfluorescent dye dihydrorhodamine 123 to fluorescent rhodamine 123 by radical species released into the media. To identify optimal conditions for expression of tissue factor, mononuclear cells were incubated for 5h in RPMI containing different concentrations of heat-inactivated FBS (HI-FBS), and LPS, zymosan, PGN or PMA as stimuli. Expression of tissue factor was determined using a one-stage recalcification assay performed in an automated nephelometric coagulation analyzer. Neither LPS nor zymosan increased ROS production by granulocytes incubated in PBG media. In contrast, granulocytes incubated in RPMI had dose-dependent increases in ROS production in response to zymosan and PGN. ROS production was significantly increased by incubation with concentrations of LPS of 0.01microg/ml or greater, and by zymosan concentrations of 0.1microg/ml or greater. ROS production in response to incubation with PMA was significantly increased starting at 10(-7)M, and was significantly greater for cells incubated in RPMI than cells incubated in PBG. LPS-, zymosan- and PGN-stimulated procoagulant activity increased in a dose-dependent manner, whereas PMA-stimulated procoagulant activity peaked at 10(-7)M. Increasing concentrations of HI-FBS significantly increased LPS-, zymosan- and PGN-induced procoagulant activity of mononuclear cells. Results obtained in this study indicate production of ROS by canine granulocytes is optimal when these cells are incubated for 3h in RPMI with LPS (0.1microg/ml), zymosan (10 microg/ml), PGN (10 microg/ml), and PMA (10(-7)M). Furthermore, canine mononuclear cells express procoagulant activity in response to LPS, zymosan, PGN, and PMA, and responses to LPS, zymosan and PGN are enhanced by the addition of HI-FBS. These findings suggest that HI-FBS retains important serum proteins that facilitate interactions between each of these bacterial or yeast derived products and the mononuclear cells. Consequently, future studies regarding the regulation of procoagulant activity by canine mononuclear cells should be performed in the presence of HI-FBS. Both assays utilized in this study allow high throughput of samples, and therefore are appropriate choices for rapid screening of conditions and/or therapeutic interventions affecting the canine inflammatory system.     

An investigation of heavy metal exposure and risks to wildlife in the Kafue flats of Zambia

Exposure and ecological risks to heavy metals (copper, zinc, manganese, iron) at Lochnivar and Blue Lagoon National Parks in wildlife dependent on the Kafue river contaminated with mining waste was evaluated. Samples included water, fish, grasses and Kafue Lechwe (Kobus leche kafuensis) liver. At both parks copper ranged from 0.03-0.04 mg/l; 3.0-6.0 mg/kg; 11.0-44.0 mg/kg; trace -199.0 mg/kg; while zinc was 0.01 mg/l; 32.0-82.0 mg/kg; 15.0-21.0 mg/kg; and 52.0-138.0 mg/kg; in water, fish, grasses and lechwe, respectively. Manganese ranges were 0.15-0.16 mg/l; 7.0-18.0 mg/kg; 51.0-145.0 mg/kg; and 40.0-53.0 mg/kg while iron ranges were 0.13-0.14 mg/l; 26.0-134.0 mg/kg; 1766.0-1797.0 mg/kg; and 131.0-856.0 mg/kg; in water, fish, grasses and lechwe, respectively. Levels in all samples except water were high indicating potential for adverse effects.     

Effect of intramammary injection of rboGM-CSF on milk levels of chemiluminescence activity, somatic cell count, and Staphylococcus aureus count in Holstein cows with S. aureus subclinical mastitis

The effect of intramammary injection of recombinant bovine granulocyte-macrophage colony-stimulating factor (rboGM-CSF, 400 microg/10 mL) on quarter milk levels of chemiluminescence (CL) activity, and somatic cell count (SCC) and shedding pattern of Staphylococcus aureus was investigated. Ten Holstein cows, naturally infected with S. aureus were used, with either early-stage or late-stage subclinical mastitis. Injection of rboGM-CSF caused a remarkable increase in milk CL activity with a peak at 6 h after the cytokine injection in the early- and late-stage groups. In the early-stage group, milk SCC stayed around preinjection level at 6 h, rose significantly on days 1 and 2, and was followed by a smooth and significant decline to an under preinjection level (below 200 000 cells/mL) on day 7 postinjection. Alternatively, in the late-stage group, milk SCC rose significantly at 6 h after the cytokine injection and maintained high levels thereafter. The milk S. aureus count decreased drastically by the cytokine injection in the early-stage group. The bacterial count was moderately decreased in the late-stage group, but increased back to preinoculation levels on day 7 after the cytokine injection. The results suggest that the rboGM-CSF has a potential as a therapeutic agent for S. aureus infection causing subclinical mastitis of dairy cows, if the cytokine is applied at the initial stage of infection.     

Identification of the core rumen bacterial taxa and their population dynamics during the fattening period in Japanese Black cattle

The rumen microbiota comprises a vast range of bacterial taxa, which may affect the production of high-quality meat in Japanese Black cattle. The aim of this study was to identify core rumen microbiota in rumen fluid samples collected from 74 Japanese Black cattle raised under different dietary conditions using 16S rRNA gene amplicon sequencing. In the rumen of fattening Japanese Black cattle, 10 bacterial taxa, showing >1% average relative abundance and >95% prevalence, irrespective of the dietary conditions and the fattening periods, were identified as the core rumen bacterial taxa, which accounted for approximately 80% of the rumen microbiota in Japanese Black cattle. Additionally, population dynamics of the core rumen bacterial taxa revealed two distinct patterns: Prevotella spp. and unclassified Bacteroidales decreased in the mid-fattening period, whereas unclassified Clostridiales, unclassified Ruminococcaceae, Ruminococcus spp., and unclassified Christensenellaceae increased during the same period. Therefore, the present study reports the wide distribution of the core rumen bacterial taxa in Japanese Black cattle, and the complementary nature of the population dynamics of these core taxa, which may ensure stable rumen fermentation during the fattening period.     

Identification of bovine serum albumin as an IgE-reactive beef component in a dog with food hypersensitivity against beef

IgE-reactive beef components were examined by an immunoblot analysis using a serum from a dog with food hypersensitivity against beef. The immunoblot analysis revealed a distinct band at approximately 66 kDa and a faint band at approximately 50 kDa. The immunoblot analysis for serum IgE reactivity to bovine serum albumin (BSA) also revealed a positive band at 66 kDa. Serum IgE reactivity to the 66-kDa protein of beef was diminished by pre-incubating the serum sample with BSA. Furthermore, a positive reaction to BSA was detected in intradermal testing in the dog. These results clearly indicated that BSA was an IgE-reactive beef component in the dog with food hypersensitivity against beef.     

Stress responses in neonatal meat and layer Nagoya chicks

We reported that meat chicks have either a greater capability to acclimatize to novel environments, or a blunted hypothalamic‐pituitary‐adrenal response to novel environments compared with layer chicks in a commercial base. The present study compared the differences in behavior and plasma corticosterone concentrations under isolation‐induced stress between neonatal meat and layer Nagoya chicks which had been separated from the same population. Both types of neonatal chicks reared in groups were individually separated and their spontaneous activity and distress‐induced vocalizations were monitored for 10 min. The responses of the two types were remarkably different, with the meat chicks being less active than the layer chicks. Distress‐induced vocalizations were fewer in the meat than in the layer chicks. The meat chicks spent more time in a sleeping posture during isolation‐induced stress. Plasma corticosterone concentrations measured at the end of the test tended to be higher in the layer chicks than in meat ones, but not significantly. In conclusion, the selection of Nagoya chickens for meat and layer may have trends similar to those observed in commercial chickens in relation to stress susceptibility.     

Seasonal and interannual variation in net ecosystem production of an evergreen needleleaf forest in Japan

Carbon dioxide (CO₂) flux was measured above the forest at the Fujiyoshida site on the northern slope of Mount Fuji in Japan in 2000?C2008 using an eddy covariance technique. The forest mainly consists of Japanese red pine (Pinus densiflora) and Japanese holly (Ilex pedunculosa). The 9-year average of monthly mean net ecosystem production (NEP) ranged from ?0.1?g?C?m^?2?day^?1 in January to 2.5?g?C?m^?2?day^?1 in May. The maximum net uptake was observed in May, although gross primary production (GPP) was highest in July. Variation in the leaf amount did not notably affect seasonal variation in GPP. This site was characterized by carbon uptake even in winter, if the meteorological conditions were conducive for photosynthesis and a resulting long period of carbon uptake. The 9-year averages of annual NEP, GPP, and ecosystem respiration (RE) were 388, 1,802, and 1,413?g?C?m^?2?year^?1, respectively. The annual NEP was lowest in 2003 and highest in 2004 over the 9?years. Year-to-year variability of NEP mainly depended on air temperature and photosynthetically active radiation in summer, and the dependence of the deviation of annual NEP on that of GPP was greater than that of RE. Long-term observational data indicated that the carbon uptake ability at the study site was at a moderate level in comparison with other temperate humid evergreen forests around the world. These data also indicated that the site had a high carbon uptake ability compared with other deciduous forests in Japan because of the duration of carbon uptake.