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.     

Metabarcoding of feces and intestinal contents to determine carnivorous diets in red-crowned cranes in eastern Hokkaido,Japan

The red-crowned crane Grus japonensis in Hokkaido, Japan forms a closed population as a residence that is independent of the mainland population. Based on observations of a limited number of individuals as well as cranes in captivity, red-crowned cranes are omnivores and eat fish, worms, insects and plants in their own territories except in winter, when they are fed with dent corn that is supplied in eastern Hokkaido. DNA metabarcoding based on high throughput sequencing was carried out using universal primer sets for cytochrome oxidase subunit I gene. Feces from 27 chicks collected in June and July in the period from 2016 to 2018 and intestinal contents from 33 adult and subadult cranes that were found dead almost throughout year in 2006–2013 in the field in eastern Hokkaido were used. Although compositions varied considerably in the cranes, both insects and fish were found in adults and subadults to the same extents, while insects were predominant in chicks. Both insects and fish were detected in all seasons for adults and subadults. Horse flies, scarab beetles and weevils accounted for the most of the insects regardless of the life stage. Dace, stickleback, flatfish and sculpin were the major fish species in adults, while chicks ate almost only stickleback. The results provide the first comprehensive data on carnivorous diets in wild red-crowned cranes in eastern Hokkaido as basis for conservation of red-crowned cranes, for which the life style and area continue to change.     

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.     

Physiological and Mineralogical Properties of Arsenic-Induced Chlorosis in Barley Seedlings Grown Hydroponically

The experiment was carried out to investigate the effects of arsenic (As) on the physiological and mineralogical properties of barley (Hordeum vulgare L. cv. ‘Minorimugi’). The plants were grown in nutrient solution treated with 0, 6.7, 33.5, and 67 μ M As (0, 0.5, 2.5, and 5 ppm As, respectively) in the phytotron. Dry matter yield of shoots and roots decreased significantly with the As treatments, indicating that barley plants are As-sensitive and As-toxicity depends on the As concentration in the rooting medium. Necrosis in older leaves and chlorosis symptoms (whitish color) in the fully developed young leaves were observed at the 33.5 and 67 μ M As treatments. Arsenic concentration, accumulation, and translocation increased with the increase of As concentration in the rooting medium. Arsenic was mostly concentrated in roots and a little amount was moved to shoots, indicating that As was not easily translocated to shoots of barley seedlings. Concentrations and accumulations of phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), manganese (Mn), zinc (Zn), and copper (Cu) decreased significantly in shoots for 33.5 and 67 μ M As treatments as compared to the 0 μ M As treatment. Concentrations of P, K, Ca, Mg, Mn, and Cu decreased in roots, but Zn concentration increased in roots at 67 μ M As treatment. Accumulations of P, K, Ca, Mg, Mn, Zn, and Cu in roots also decreased significantly at 67 μ M As treatment. Accumulation of P and the cations showed negative relationship with As. Concentration of Fe decreased in shoots at 33.5 and 67 μ M As treatments where chlorosis was induced in the young leaf but increased in roots at 33.5 and 67 μ M As treatments. It was suggested that As might induce iron (Fe)-chlorosis in the plants. Among the micronutrients, Fe translocation was more affected than others by As. Phytosiderophore (PS) accumulation in roots, which is a symptom of Fe-deficiency in grasses, did not change significantly between 0 and 33.5 μ M As treatments; indicating that As-induced chlorosis did not enhance PS accumulation in roots and decreased due to As-toxicity at 67 μ M As treatment.