Improvement of an enzyme-linked immunosorbent assay for equine herpesvirus type 4 by using a synthetic-peptide 24-mer repeat sequence of glycoprotein G as an antigen

To increase the sensitivity of an enzyme-linked immunosorbent assay (ELISA) for equine herpesvirus type 4 (EHV-4) that uses a 12-mer peptide of glycoprotein G (gG4-12-mer: MKNNPIYSEGSL) [4], we used a longer peptide consisting of a 24-mer repeat sequence (gG4-24-mer: MKNNPIYSEGSLMLNVQHDDSIHT) as an antigen. Sera of horses experimentally infected with EHV-4 reacted much more strongly to the gG4-24-mer peptide than to the gG4-12-mer peptide. We used peptide ELISAs to test paired sera from horses naturally infected with EHV-4 (n=40). gG4-24-mer ELISA detected 37 positive samples (92.5%), whereas gG4-12-mer ELISA detected only 28 (70.0%). gG4-24-mer ELISA was much more sensitive than gG4-12-mer ELISA.     

Effects of pre-feeding of a corn silage-based supplement on the feed intake, milk production and nitrogen utilization of grazing dairy cows

Four Holstein cows were used to determine the effect of timing of the feeding of a corn silage (CS)‐based supplement on the feed intake, milk production and nitrogen utilization of grazing dairy cows. The cows were fed the supplement 2 h before grazing (pre‐grazing) or immediately after grazing (post‐grazing). Cows were grazed for 5 h per day under a rotational grazing system. There was no difference in the herbage and total feed intake between treatments. The milk protein yield for pre‐grazing tended to be higher than that for post‐grazing, whereas the milk yield did not differ between treatments. The total nitrogen intake for pre‐grazing tended to be higher than that for post‐grazing (P = 0.06). There was no difference in the urinary nitrogen output between treatments, whereas the proportion of urinary nitrogen output : total nitrogen intake for pre‐grazing tended to be lower than that for post‐grazing (P = 0.06). The milk nitrogen output and nitrogen retention for pre‐grazing tended to be higher than that for post‐grazing (milk nitrogen, P = 0.06; nitrogen retention, P = 0.05). Nitrogen utilization of grazing dairy cows was improved by feeding a CS‐based supplement before grazing.     

Identification of marine algicidal Flavobacterium sp. 5 N-3 using multiple probes and whole-cell hybridization

ABSTRACT: We designed five 16S rRNA-targeted oligonucleotide probes (Sp probes) specific for Flavobacterium sp. 5 N-3, which inhibits the growth of red tide phytoplankton Gymnodinium mikimotoi (Dinophyceae). These probes were evaluated by whole-cell hybridization against 5 N-3 cells incubated under laboratory conditions. The fluorescence signal from the cell detected with Sp probe mix5, a mixture of the five probes, was 8.4-fold higher than that obtained with only one Sp probe (Sp01RF). The signal obtained by this method was strong enough to recognize 5 N-3 cells and count them under the epifluorescence microscope, while the signal was often undetectable when a single probe was used. Fluorescence intensities of cells at stationary phases and of 'starved' cells in sterile seawater using Sp probe mix5 were low but still sufficient for enumeration. These Sp probes did not hybridize with 11 strains from the Cytophaga/Flavobacteria/Bacteroides phylum and did react with strain 5 N-3 following whole-cell hybridization. These results show that 5 N-3 cells cultivated under our laboratory conditions can be detected by whole-cell hybridization with the five designed probes. These data also suggest that this technique may be useful for detection of an algicidal bacterium 5 N-3 in the natural environment.     

Influence of Equine Conformation on Rider Oscillation and Evaluation of Horses for Therapeutic Riding

To obtain basic knowledge about selecting horses for therapeutic riding, the influence of equine conformation on rider oscillation and relationships between these factors and the evaluation on horses as the therapeutic riding were studied. Thirty-five riding horses were used. Equine conformation was estimated by 24 indices. Rider oscillation was measured by an accelerometer fixed at the rider’s waist. The spatial position of the oscillation was estimated by a double integration of the acceleration. Horses were evaluated for therapeutic riding by a Riding for the Disabled Association instructor as a rider. Evaluations were on a scale of 1 to 5, with 5 being the highest score for 27 items. Horses were classified into 4 groups: the short and narrow (SN), short and wide (SW), tall and narrow (TN), and tall and wide (TW). The frequencies of rider oscillation both at walk and trot were higher (P<0.01), and the vertical (P<0.01) and longitudinal (P<0.05) amplitudes at trot were smaller, on short horses than on tall horses. The vertical amplitude at walk was smaller (P<0.05) and the lateral amplitude at trot was larger (P<0.01) on wide horses than on narrow horses. Short horses could be used for the rider who requires side walkers. Wide horses could be used for relieving muscular tension and for the rider who could not maintain good balance on the horse. Short and wide horses should be suitable for therapeutic riding.     

Mean retention time of digesta in the different segments of the equine hindgut

Six Thoroughbred horses were used to determine the mean retention time (MRT) of digesta in the different segments of the hindgut. The horses were fed timothy hay or silage in equal amounts (1.6% bodyweight [BW]/day) every 3 h/day. Hay or silage labeled with the rare earth elements cerium, dysprosium, neodymium, ytterbium, lanthanum, samarium and praseodymium, were fed to the horses 36, 30, 24, 18, 12, 6 and 3 h before slaughter, respectively. Just after slaughter, digesta samples in the different segments were collected and determined for the contents of the rare earth elements. Using these data, a method to calculate the MRT in the different segments was proposed. There were no significant differences between diets in the MRT of each segment. The averaged MRT of hay and silage in the cecum, right ventral colon, left ventral colon, left dorsal colon, right dorsal colon and small colon was 2.9, 3.1, 5.9, 1.0, 4.0, and 4.0 h, respectively. The dry matter weight of the digesta was related to the MRT in the right ventral colon (r = 0.94, P = 0.005), left ventral colon (r = 0.87, P = 0.03), left dorsal colon (r = 0.98, P < 0.001) and right dorsal colon (r = 0.95, P = 0.004), but it was not related to the MRT in the cecum and small colon.     

Association of BoLA-DRB3 alleles identified by a sequence-based typing method with mastitis pathogens in Japanese Holstein cows

The association of the polymorphism of bovine leukocyte antigen ( BoLA-DRB3 ) genes identified by the polymerase chain reaction sequence-based typing (PCR-SBT) method with resistance and susceptibility to mastitis caused by pathogenic bacteria was investigated. Blood samples for DNA extraction were collected from 194 Holstein cows (41 healthy cows and 153 mastitis cows including 24 mixed-infection cows infected with 2 or 3 species of pathogens) from 5 districts of Chiba prefecture, Japan. Sixteen BoLA-DRB3 alleles were detected. The 4 main alleles of DRB3*0101 , *1501 , *1201 , and *1101 constituted 56.8% of the total number of alleles detected. Mastitis cows were divided into 2 groups: group 1 with single-infection cows and group 2 with all mastitis cows including 24 mixed-infection cows. The differences in the frequencies of BoLA-DRB3 alleles and the number of cows homozygous or heterozygous for each BoLA-DRB3 allele between healthy cows and the 2 groups of mastitis cows were evaluated. Furthermore, similar comparisons were performed between healthy cows and the 2 groups of mastitis cows for each mastitis pathogen. It was considered that the 4 alleles, namely, DRB3*0101 , *1501 , *1201 , and *1101 had specific resistance and susceptibility to 4 different mastitis pathogens. Thus, DRB3*0101 might be associated with susceptibility to coagulase-negative Staphylococci and Escherichia coli , and DRB3*1501 might be associated with susceptibility to Escherichia coli . However, DRB3*1101 might be associated with resistance to Streptococci and coagulase-negative Staphylococci , and DRB3*1201 , with resistance to Streptococci , Escherichia coli , and Staphylococcus aureus .     

Mobiloization of intracellular calcium ions in chicken and rat lymphocytes induced by T cell mitogens

Cytosolic Ca²⁺ is known to be an important factor in intracellular signaling pathways that regulate several cellular functions. The present study was designed to measure the intracellular concentrations of Ca²⁺ ([Ca²⁺]_i) in T cell mitogen-stimulated chicken lymphocytes, and to compare the results with those in rat lymphocytes. [Ca²⁺]_i was increased in the thymocytes, splenocytes and bursacytes of chickens, and in the thymocytes and splenocytes of rats following exposure to the mitogens phytohaemagglutinin (PHA) and concanavalin A (ConA). Increases were greatest in the thymocytes followed by the splenocytes and bursacytes. The PHA-induced changes in the thymocytes and splenocytes were similar in chickens and rats, but the ConA-induced increases were significantly lower in the chickens than rats. Pretreatment with EGTA before the application of PHA and ConA completely suppressed the rise in [Ca²⁺]_i in all the chicken lymphocytes, indicating that the increases that occurred in PHA- and ConA-treated chicken lymphocytes could be entirely attributed to the influx of extracellular Ca²⁺. On the other hand, the PHA- and ConA-induced increase in [Ca²⁺]_i in rat lymphocytes was not completely suppressed by EGTA, indicating the recruitment of Ca²⁺ from the intracellular Ca²⁺ pool. The results suggest species differences in the Ca²⁺-based responses to T cell mitogens between chicken lymphocytes and rat lymphocytes.     

Response to mitogens of chicken splenocytes determined by three methods of measurement

The aim of the present study was to examine whether the 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyl tetrazolium bromide (MTT) colorimetric assay can be applied to measurement of mitogen‐induced chicken splenocyte activation. Activation was also measured by a ³H‐thymidine uptake assay and a viable cell count assay. Optimal concentrations of mitogens and incubation periods required for maximal responses to mitogens differed between the MTT assay and the viable cell count and thymidine uptake assays. This probably reflects differences in the activities measured by the MTT assay which detects mitochondrial enzyme activity, and the thymidine uptake and viable cell count assays which detect cellular proliferation activity. The validity of the MTT assay was supported by the observation that the mitogen‐induced increase in succinate dehydrogenase activity paralleled the level of mitogen‐induced MTT formazan production. Mitogen concentrations inducing maximal formazan formation in chicken splenocytes were higher than those for chicken peripheral blood lymphocytes reported previously. Results of the present study indicate that mitogen‐induced chicken splenocyte activation could be measured by the MTT colorimetric assay, although mitogen concentrations and incubation periods required for maximal splenocyte activation differed between the MTT assay and the other two assays used in this study.     

Effect of feeding non-fibrous carbohydrate before grazing on intake and nitrogen utilization in dairy cows throughout the grazing season

Ten lactating cows were used to determine the effect of feeding non-fibrous carbohydrate (NFC) supplement before grazing on feed intake and nitrogen (N) utilization throughout a grazing season. The experiment was conducted from June to September. Cows grazed twice a day (2.5 h × 2) under a set stocking system and were fed NFC supplement (1 kg/4 kg of milk yield) 2 h before grazing (PRE) or immediately after grazing (POST). Cows were also fed a grass and corn silage mixture ad libitum. Herbage dry matter intake (DMI) was greater for PRE than for POST throughout the experiment and decreased from June to September. Conversely, silage DMI was less for PRE than for POST throughout the experiment and increased over the grazing season. Consequently, total DMI for PRE did not differ from that for POST. Milk urea-N concentration and urinary urea-N excretion in June did not differ between the treatments, whereas that after July was higher for PRE than for POST. Proportion of urinary N excretion to absorbed N intake in June was lower for PRE than for POST, but that after July was higher for PRE than for POST. Feeding NFC supplement before grazing would improve N utilization when cows eat large amounts of herbage high in N.