Genetic Structure of Populations of the Pinewood Nematode Bursaphelenchus xylophilus, the Pathogen of Pine Wilt Disease, Between and Within Pine Forests

ABSTRACT We analyzed the genetic structure of Bursaphelenchus xylophilus populations within individual trees (subpopulations) in three distant pine forests (Tanashi, Tsukuba, and Chiba in Japan) based on the polymorphism of four microsatellite (SSR) markers. Most of the nematodes from subpopulations in Tanashi showed the same genotype over 2 years, indicating that nematodes of that genotype dominated there for years. In contrast, 16 and 15 genotypes were identified in nematode populations from Tsukuba and Chiba, respectively. Despite the high genetic diversity within the Tsukuba and Chiba populations, extremely low genetic diversity was observed within the subpopulations. The genetic difference between the Tsukuba and Chiba populations was significantly smaller than that between Tanashi and either Tsukuba or Chiba. Observed heterozygosity was significantly less than expected based on Hardy-Weinberg equilibrium. These findings are best explained by a founder effect, geographic isolation between populations, explosive nematode multiplication from a small number within individual trees, and the Wahlund effect.     

Effects of a combination of hinokitiol (β‐thujaplicin) and an organic acid mixture on ruminal fermentation in heifers fed a high‐grain diet

This study evaluated the effects of hinokitiol (a natural antibacterial compound extracted from Thujopsis dolabrata var. hondai) and an organic acid mixture (citrate content 50%) on ruminal fermentation. Antibacterial properties were examined by measuring minimal inhibitory concentration. Hinokitiol at 1.56 µg/mL or an organic acid mixture at 1600 µg/mL inhibited Streptococcus bovis growth. The combination of 0.78 µg/mL hinokitiol and 200 µg/mL of an organic acid mixture also inhibited S. bovis growth. Both hinokitiol and the hinokitiol and an organic acid mixture combination showed strong antibacterial properties on Gram‐positive bacteria such as S. bovis, but relatively weak antibacterial activities on Gram‐negative bacteria such as Megasphaera elsdenii. Three ruminally cannulated heifers were fed a bloat‐producing diet containing barley, pelleted alfalfa meal, soybean meal and salt without long‐cut roughage to investigate the ruminal characteristics in vivo. Feeding to heifers a bloat‐producing diet containing 7.8 mg/kg hinokitiol and 0.2% of an organic acid mixture significantly decreased the increase in stable ingesta volume. Hinokitiol or an organic acid mixture did not affect ruminal volatile fatty acids, protozoa and bacteria. These results suggest that a combination of hinokitiol and an organic acid mixture might reduce frothy bloat in cattle fed high‐grain diets.     

Effects of daily management changes on behavioral patterns of a solitary female African elephant (Loxodonta africana) in a zoo

This study investigated the effects of changes in daily management on behavior of a solitary female elephant in a zoo. The activity budget and space utilization of the subject and the management changes were recorded for 1 year after the conspecific male died. The observation days could be categorized into five clusters (C1–C5) by the characteristic behavioral pattern of each day. C1 had the highest percentage of resting of all clusters, and was observed after the loss of the conspecific and the beginning of use of the indoor exhibition room at night. C2, which had the highest percentage of stereotypy of any cluster, was observed after the beginning of habituation to the indoor exhibition room. Also, when the time schedule of management was changed irregularly, the subject frequently exhibited stereotypic pacing (C2, C4). The subject tended to rest when exhibiting lameness in the left hind limb (C3). In C5, activity reached a high level when she could utilize a familiar place under a stable management schedule. These results indicate that management changes affected the mental stability of an elephant in the early stage of social isolation.     

Utilization of flow cytometry for festulolium breeding (Lolium multiflorum (2x) × Festuca arundinacea (6x))

Festulolium is a hybrid between Festuca and Lolium species that has valuable agronomic traits from both grass species. The purpose of our breeding program is to produce hexaploid festulolium that introduces tolerance to summer depression into Italian ryegrass (Lolium multiflorum) by crossing it with tall fescue (Festuca arundinacea). However, we found the DNA ploidy of hexaploids was not stable and was reduced in successive generations. We aimed to find out how to obtain stable high-ploidy festulolium. F1 hybrids of L. multiflorum and F. arundinacea were produced. The F3 generation was produced from putative hexaploid F2 individuals by open pollination. The F4 to F6 generations were obtained by polycrossing. The DNA ploidy levels of F2 to F6 individuals were estimated by flow cytometry. Cytological characteristics of the F5 and F6 individuals were investigated by FISH and GISH. The DNA ploidy level of hexaploid festulolium was reduced and stabilized at almost the same level as a tetraploid. Seed fertility was inversely correlated with an increase in ploidy level. GISH revealed no preferential Lolium transmission. FISH with a telomere probe revealed that counting the exact number of chromosomes in festulolium was difficult. DNA ploidy level was strongly correlated with the number of chromosomes.     

Bacillus cereus from the environment is genetically related to the highly pathogenic B. cereus in Zambia

To follow-up anthrax in Zambia since the outbreak in 2011, we have collected samples from the environment and the carcasses of anthrax-suspected animals, and have tried to isolate Bacillus anthracis. In the process of identification of B. anthracis, we collected two isolates, of which colonies were similar to B. anthracis; however, from the results of identification using the molecular-based methods, two isolates were genetically related to the highly pathogenic B. cereus, of which clinical manifestation is severe and fatal (e.g., pneumonia). In this study, we showed the existence of bacteria suspected to be highly pathogenic B. cereus in Zambia, indicating the possibility of an outbreak caused by highly pathogenic B. cereus.     

Correlation of minimum inhibitory concentrations between human and animal antimicrobials against Escherichia coli isolated from livestock

We analyzed the correlation between minimum inhibitory concentrations (MICs) of antimicrobials used in humans and those used in animals to enable comparison of antimicrobial susceptibility between Escherichia coli isolated from humans and those from animals. We compared the following pairs of MIC data: piperacillin (PIPC) to ampicillin (ABPC), amikacin (AMK) to kanamycin (KM), minocycline (MINO) to oxytetracycline (OTC), and levofloxacin (LVFX) to enrofloxacin (ERFX) using 103 isolates of E. coli from healthy livestock (cattle, pigs, broiler chickens, and layer chickens). Kappa analysis of the agreement for resistance and susceptibility between PIPC and ABPC, AMK and KM, MINO and OTC, and LVFX and ERFX showed almost perfect (κ = 0.81), slight (κ = 0.12), fair (κ = 0.37), and moderate (κ = 0.46) agreement, respectively. Within the antimicrobial pairs, all isolates resistant to the human antimicrobial were also resistant to the veterinary antimicrobial. However, there was less agreement within the pairs for those isolates that were sensitive to the human antimicrobial. The percentage agreement for susceptibility, defined as the percentage of isolates sensitive to both antimicrobials compared with isolates sensitive to both antimicrobials, as well as those sensitive only to the human antimicrobial, was 89.9%, 87.3%, 64.0%, and 89.9% for PIPC and ABPC, AMK and KM, MINO and OTC, and LVFX and ERFX, respectively. Our results suggest that the possibility of missing the resistance for antimicrobials used in human medicine by examining MICs for the equivalent antimicrobials used in veterinary medicine is low.     

Effect of feeding Bacillus subtilis on rumen fermentation,blood metabolites,nutrient digestibility,and energy and nitrogen balances in non-lactating crossbred cows

The purpose of this study was to investigate the effects of feeding Bacillus subtilis on rumen fermentation, blood metabolites, nutrient digestibility, and energy and nitrogen balances in non-lactating crossbred (Holstein-Friesian × Bos indicus) cows. Four cows were assigned to the control and B. subtilis diets in a crossover design, and respiratory and metabolic experiments were conducted. For the B. subtilis diet, B. subtilis DSM15544 spores were added at the rate of 1.0 × 10¹⁰ CFU/head/day to the control diet. At 4 hr after feeding, cows fed the B. subtilis diet had increased levels of i-butyric acid in the rumen fluid and tended to have lower concentrations of plasma non-esterified fatty acids when compared with cows fed the control diet. This suggests that feeding B. subtilis could improve energy efficiency. However, there was no effect on energy retention in this study. Although there were no effects on nutrient digestibility, nitrogen balance, or methane production, heat production was significantly higher in cows fed the B. subtilis diet than in those fed the control diet.     

Performance of Pot-Grown Seedlings of the Dipterocarp Dryobalanops lanceolata with Controlled-Release Fertilizer after Transplantation to the Shifting Cultivation Land in Sarawak, Malaysia

In the present study, the focus was placed on the survival, growth, nutrient status, and ectomycorrhizal formation of pot-grown dipterocarps treated with a controlled-release fertilizer or a chemical fertilizer when transplanted to abandoned shifting cultivation land. Either a chemical fertilizer (CF) or a 700-day controlled-release fertilizer (CRF) had been applied to Dryobalanops lanceolata seedlings at the rates of 0, 2, 5, and 10 g fertilizers per pot in the nursery of the Forest Research Station Niah (FRSN), Miri, Sarawak, Malaysia. The seedlings were cultivated for 18 months in the nursery, and then transplanted to abandoned shifting cultivation land around the FRSN. At 16 months after transplantation, the plants in the CRF treatments showed a good performance in terms of growth and survival ability, because of the sufficient amounts of nutrients accumulated in plants with a sound shoot form and high photosynthetic activity at the nursery stage. On the other hand, many saplings in the CF treatments at the rates of 5 g and 10 g died in the field due to their spindly shoot form and low photosynthetic activity in the nursery, even though the saplings that survived showed on adequate growth. Therefore, the application of slow-release type CRF in the nursery was considered to be feasible and suitable for successful establishment of a dipterocarp plantation in the field. Ectomycorrhizal fungus present in the nursery would sustain and be correlated with P uptake of the saplings after transplantation.