Induction of a cross-reactive antibody response to influenza virus M2 antigen in pigs by using a Sendai virus vector

Protecting pigs from simultaneous infection with avian, swine, and human influenza viruses would be an effective strategy to prevent the emergence of reassortants with pandemic potential. M2 protein is a candidate antigen for so-called 'universal vaccines,' which confer cross-protection to different influenza viruses in a strain- and subtype-independent manner. We tested whether a recombinant F gene-deleted Sendai virus vector that contained an M2 gene derived from an H5N1 avian influenza virus (SeV/ΔF/H5N1M2) could induce a cross-reactive antibody response to the extracellular domain of M2 protein (M2e) in pigs. SeV/ΔF/H5N1M2 induced an antibody response to M2e when the vector was inoculated intramuscularly. The antibodies induced by SeV/ΔF/H5N1M2 cross-reacted with M2e derived from different avian, swine, and human influenza viruses. In mice, however, SeV/ΔF/H5N1M2 did not confer cross-protection to challenge with a heterologous H3N2 influenza virus. Our results confirm those of other groups indicating that antibodies to M2e do not mediate protection to influenza viruses in pigs.     

Trapped neutrophil syndrome in a Border Collie dog: clinical, clinico-pathologic, and molecular findings

Trapped neutrophil syndrome (TNS) is an autosomal recessive inherited neutropenia known in Border Collies since the 1990's. Recently, the causative mutation has been identified in the canine VPS13B gene and a DNA-based diagnosis has now become available. The present paper describes clinical and clinico-pathologic findings in a Border Collie with TNS that was molecularly diagnosed for the first time in Japan. In a 10-week-old male Border Collie with microgenesis and symptoms related to recurrent infections, a hematological examination revealed severe leukopenia due to neutropenia, suggesting the dog to be affected by inherited neutropenic immunodeficiency. Direct DNA sequencing demonstrated that the dog was homozygous for the causative mutation of TNS and both its parents were heterozygous carriers. In addition, a simple and rapid polymerase chain reaction-based length polymorphism analysis coupled with microchip electrophoresis was developed for the genotyping of TNS. This assay could discriminate clearly all genotypes, suggesting that it was suitable for both individual diagnosis and large-scale surveys for prevention.     

Normal calves produced after transfer of embryos cultured in a chemically defined medium supplemented with epidermal growth factor and insulin-like growth factor I following ovum pick up and in vitro fertilization in Japanese black cows

The objective of this study was to examine whether high concentrations of epidermal growth factor (EGF) and/or insulin-like growth factor I (IGF-I) would have a beneficial effect on bovine embryo development in vitro and to obtain normal calves by using an ovum pick up method and embryo culture in a chemically defined medium. When compared with controls, EGF (100 or 200 ng/ml) or IGF-I (50 or 100 ng/ml) significantly increased the rate of embryos that developed into blastocysts during an 8-day culture after the in vitro fertilization of oocytes obtained from ovaries from a slaughterhouse. IGF-I induced a dose-dependent increase in cell number in both the inner cell mass and the trophectoderm, whereas EGF stimulated proliferation only in the inner cell mass. A combination of EGF (100 ng/ml) and IGF-I (50 ng/ml) produced an additive effect, and embryos developed into blastocysts at a comparatively high rate (27.9%) compared with controls (12.0%). A similar rate of development was achieved using a combination of EGF and IGF-I in the culture of embryos following ovum pick up by ultrasound-guided transvaginal follicular aspiration and in vitro fertilization, and 5 blastocysts that developed after the culture were transferred into uteri; two embryos implanted, and normal calves were born. These results suggest that the combined use of EGF and IGF-I makes bovine embryo culture in a chemically defined medium a practical and useful procedure for producing blastocysts, and its application to embryo culture following ovum pick up and in vitro fertilization could be useful for producing normal calves.     

Genetic diversity of Pinus densiflora pollen flowing over fragmented populations during a mating season

We attempted to evaluate the genetic diversity of long-distance transported pollen flowing over fragmented Pinus densiflora populations during a mating season. A P.?densiflora clonal seed orchard, which was located in a rural area where many fragmented populations exist, was selected for pollen capture. Immigrant pollen captured by three clones having different flowering times was regarded as the pollen flowing over fragmented populations during a mating season. The genetic diversity (H _e) values of the immigrant pollen captured by the three clones were high (H _e?>?0.894). The correlation of paternity (r _p) values of the seeds having immigrant parent generated from the three clones were calculated to be negative. From these parameters, the pollen cloud is considered to have maintained high genetic diversity during the mating season. The genetic composition of the pollen cloud showed slight variation. The pollen captured by different trees (i.e., clonal ramets of the three clones) was significantly different based on analysis of molecular variance. Especially, the pollen pools captured by trees planted in the western side of the orchard were significantly different from the gene pool of the surrounding populations. Factors affecting this differentiation could be that the donors of the pollen transported to the orchard vary with time, as well as nonuniform dispersal of the pollen. From these results, the pollen flowing over fragmented P.?densiflora populations is considered to have high genetic diversity, compensating to some extent for fragmentation.     

Seasonal variations in the incidence of pine wilt and infestation by its vector, Monochamus alternatus, near the northern limit of the disease in Japan

During the last few decades, pine wilt disease has spread to cool-climate regions in Japan and, more recently, the potential risk of it spreading into the European midwest has also become a concern. In a coastal pine stand (84.7?ha) in Akita, near the northern limit of pine wilt disease in Japan, we investigated seasonal variations in the incidence of damage caused by the disease to trees and oviposition by the disease’s insect vector, Monochamus alternatus, during a two-year period. Foliage discoloration occurred throughout each year, and its seasonal variation showed a bimodal pattern in Pinus thunbergii (a higher peak in May–June and a smaller peak in October) and a clear peak in June in P. densiflora, which differed from the patterns in seasonal variation seen for warm-climate regions. Oviposition scars by M. alternatus were found in 40–45% of the trees damaged each year. The percentage of trees that had oviposition scars was higher in P. thunbergii than in P. densiflora. This appeared to reflect the difference in seasonal discoloration pattern between the two species. Analysis of the oviposition risk showed that trees that exhibited discoloration starting between July and October had a significantly higher risk or significantly higher oviposition scar densities, particularly for those that became discolored between August and September (2.5–14.6-fold higher risk than during other months). Oviposition scar densities per damaged tree were similar within the period of higher oviposition risk. Considering both oviposition risks and scar densities, we concluded that trees with discoloration that become apparent between July and October are important targets for preventing the spread of pine wilt disease in Akita.     

The Negative Relationship between Fouling Organisms and the Content of Eicosapentaenoic Acid and Docosahexaenoic Acid in Cultivated Pacific Oysters,Crassostrea gigas

Bivalves serve as an important aquaculture product, as they are the source of essential fatty acids, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), in our diet. However, their cultivation in the wild can be affected by fouling organisms that, in turn, affect their EPA and DHA content. The effects of fouling organisms on the EPA and DHA contents of cultivated bivalves have not been well documented. We examined the effects of fouling organisms on the EPA and DHA contents and condition index of cultured oysters, Crassostrea gigas, in an aquaculture system. We sampled two-year-old oysters from five sites in Shizugawa Bay, Japan, in August 2014. Most of the fouling organisms were sponges, macroalgae, and Mytilus galloprovincialis. A significant negative relationship existed between the DHA content in C. gigas and the presence of sponges and macroalgae. A lower C. gigas EPA content corresponded to a higher M. galloprovincialis fouling mass and a lower C. gigas condition index. This can be explained by dietary competition between C. gigas and M. galloprovincialis for diatoms, which were the main producer of EPA in our study sites. Our findings indicate that fouling organisms likely reduce the EPA and DHA content in cultivated oysters. Therefore, our results suggest that the current efforts to remove fouling organisms from oyster clusters is an effective strategy to enhance the content of EPA and DHA in oysters.     

Genetic and enzymatic analyses of metalloprotease (aureolysin) from Staphylococcus aureus isolated from domestic animals.

The gene (aur) encoding the metalloprotease (aureolysin) of Staphylococcus aureus from domestic animals was analyzed by polymerase chain reaction (PCR), PCR-restriction fragment length polymorphism (PCR-RFLP) and sequencing. The aur gene was detected in all 74 isolates from cows, pigs and chickens by PCR amplification and was classified into types I and II by PCR-RFLP patterns. The type II aur gene was contained in 36 (94.7%) of 38 protease-positive isolates as judged by skim milk agar plate culture, while type I was contained in 28 (77.8%) of 36 protease-negative isolates. The deduced amino acid sequences of aureolysins from type I and II isolates were almost identical with those of the published data. Subsequently, the two aureolysins were purified from the culture supernatants of type I and II isolates. The molecular weights of purified type I and II aureolysins were both estimated at 34kDa by SDS-polyacrylamide gel electrophoresis. These aureolysins had maximum proteolytic activity at 30-50 degrees C and pH 7.0-8.0. Their activity was inhibited by metal- and zinc-specific inhibitors, such as EDTA, EGTA and 1,10-phenanthroline. Specific activity (activity/protein) of type II aureolysin was two times higher than that of type I. These results indicated that the aur gene is highly conserved with two allelic forms (types I and II) among bovine, porcine and avian isolates of S. aureus.     

Influence of precipitations and sunshine hours on yield of paddy field grown wheat (Triticum aestivum L.) in Northern Kyushu,Japan

ABSTRACT

Relationships between wheat (Triticum æstivum L.) yield components and growing season weather conditions (e.g. temperature, precipitation, and sunshine duration) were investigated for a rice-wheat rotation paddy system situated in Northern Kyushu, Japan over a 17-year period (2000–2016). A 1.0 mm increase in precipitation from 21 to 32 days after sowing decreased wheat yield by about 27 kg ha^?1 at early seedling stage with one to two leaves (Zadoks (Z) growth stage Z1.1–1.2). Number of tillers and spikes showed significant negative correlations to the amount of precipitation during the period. An hour increase in mean daily sunshine hours from 94 to 111 days after sowing resulted in an 328 kg ha^?1 rise in grain yield at stem elongation stage with the first and second node detection (Z3.1–3.2). The grain number per square meter also showed strong positive correlations to sunshine hours during the same period. However, this positive effect of sunshine was negated when precipitation exceeded about 30 mm during the early seedling stage. Consequently, precipitations at early seedling stage and sunshine hours at stem elongation stage mostly determined wheat yield in Northern Kyushu paddy field. Increasing precipitations during the early seedling stage highlights the pressing need for effective paddy field drainage management from wheat sowing through harvest.

Abbreviations: CP: Chikushi Plains; FARC: Fukuoka prefectural Agriculture and Forestry Research Center; SARC: Saga prefectural Agricultural Research Center; DAS: days after sowing     

Feed intake,digestibility, nitrogen utilization,ruminal condition and blood metabolites in wethers fed ground bamboo pellets cultured with white‐rot fungus (Ceriporiopsis subvermispora) and mixed with soybean curd residue and soy sauce cake

Three types of bamboo pellets as a ruminant feed: P1 (ground bamboo (GB) cultured with the fungus Ceriporiopsis subvermispora (CGB) : soybean curd residue (T) : soy sauce cake (S) in a 5:4:1 ratio on a dry matter (DM) basis); P2 (GB : T : S = 5:4:1 on a DM basis); and P3 (CGB : T : S = 5.5:0.8:3.7 on a DM basis) were prepared. Four wethers were assigned in a 4 × 4 Latin square design experiment to evaluate the applicability of the bamboo pellets. The experimental treatments were C (control): fed alfalfa hay cubes (AC) only, and T1, T2 and T3: fed P1, P2, and P3 with AC by 1:1 on a DM basis, respectively. The digestibility of the DM, organic matter and acid detergent fiber of P1 were significantly higher than those of P2 and P3 (P < 0.05). The total digestible nutrient (TDN) contents of AC, P1, P2 and P3 were 56.5%, 60.2%, 53.2% and 47.0%, respectively. No significant differences in nitrogen retention or ruminal pH and NH₃ were observed among the treatment groups. The results indicate that bamboo pellets cultured with C. subvermispora and mainly mixed with soybean curd residue improved nutritional quality of ground bamboo because of its high digestibility and TDN content.     

Intraocular vaccination with an inactivated highly pathogenic avian influenza virus induces protective antibody responses in chickens

Because it is expected to induce cross-reactive serum and mucosal antibody responses, mucosal vaccination against highly pathogenic avian influenza (HPAI) is potentially superior to conventional parenteral vaccination. Here, we tested whether intraocular vaccination with an inactivated AI virus induced protective antibody responses in chickens. Chickens were inoculated intraocularly twice with 10⁴ hemagglutination units of an inactivated H5N1 HPAI virus. Four weeks after the second vaccination, the chickens were challenged with a lethal dose of the homologous H5N1 HPAI virus. Results showed that most of the vaccinated chickens mounted positive antibody responses. The median serum hemagglutination inhibition titer was 1:80. Addition of CpG oligodeoxynucleotide 2006 or cholera toxin to the vaccine did not enhance serum antibody titers. Cross-reactive anti-hemagglutinin IgG, but not IgA, was detected in oropharyngeal secretions. In accordance with these antibody results, most vaccinated chickens survived a lethal challenge with the H5N1 HPAI virus and did not shed the challenge virus in respiratory or digestive tract secretions. Our results show that intraocular vaccination with an inactivated AI virus induces not only systemic but also mucosal antibody responses and confers protection against HPAI in chickens.