Inclusion of Bacillus amyloliquefaciens strain TOA5001 in the diet of broilers suppresses the symptoms of coccidiosis by modulating intestinal microbiota

Coccidiosis is an intestinal parasitic infection and one of the most prevalent and economically damaging diseases of chickens. Furthermore, coccidia‐induced mucogenesis promotes secondary colonization by Clostridium perfringens, a major pathogen of chickens that causes necrotic enteritis. Our previous work found that supernatant of a culture of Bacillus amyloliquefaciens strain TOA5001 (BA) inhibited the growth of C. perfringens on Gifu anaerobic broth medium. Accordingly, we evaluated the effectiveness of dietary BA administration in inhibiting C. perfringens colonization of the intestine in broilers that were experimentally infected with coccidia. Ten healthy broilers from a BA‐supplemented (2 × 10⁵ colony‐forming units/g of feed) broiler group and 10 from a non‐treated group were challenged with Eimeria tenella and E. maxima (5000 oocysts of each species/chick) at 28 days old. At 36 days old, five chicks from each group were slaughtered, whereas the remaining five in each group were killed at 49 days old. Dietary BA administration into Eimeria‐challenged birds reduced coccidial symptoms such as intestinal lesions. It also modified the cecal microbiota through suppressing C. perfringens and E. coli colonization, and inducing domination of Faecalibacterium prausnitzii, the Lactobacillus group and unknown Lachnospiraceae genera by bacterial DNA‐based metagenome analyses. B. amyloliquefaciens TOA5001 supplementation suppressed the symptoms of coccidiosis by modulating cecal microbiota in Eimeria‐challenged broilers.     

Isolation and characterization of antioxidant protein fractions from melinjo (Gnetum gnemon) seeds

The protein from the seeds of melinjo ( Gnetum gnemon ) was purified using a precipitation method and ion exchange chromatographic techniques to identify the potent antioxidant and free radical scavenging activities. Two antioxidant protein fractions were isolated from G. gnemon seed with molecular weights of approximately 30 kDa (Gg-AOPI) and 12 kDa (Gg-AOPII) by SDS-PAGE. The N-terminal amino acid sequence of Gg-AOPII is Gly-Asn-Gly-Lys-Ala-Thr-Val-Ala-Ile-Leu-Val-Lys-Glu-Lys-Val-Glu-Tyr-Gly-Glu-Glu, and the result of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis showed that they were distinct from each other; no protein in database matching was found to both Gg-AOPI and Gg-AOPII. The antioxidant or free radical scavenging activities of Gg-AOPs were investigated by employing in vitro assay systems including the inhibition of linoleic acid autoxidation, scavenging effect on α,α-diphenyl-β-picrylhydrazyl free radical (DPPH), 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), reducing power, chelating abilities of metal ions Cu(2+) and Fe(2+), and protections against hydroxyl radical-mediated DNA damages. The result showed that two protein fractions exhibited significant (p < 0.05) antioxidant activities against free radicals such as DPPH, ABTS, and superoxide anion and showed activities similar to those of glutathione (G-SH) and BHT in a linoleic acid emulsion assay system. Moreover, Gg-AOPI and Gg-AOPII also exhibited notable reducing power and strong chelating effect on Fe(2+) and protected hydroxyl radical induced oxidative DNA damage. The data obtained by the in vitro systems obviously established the antioxidant potency of Gg-AOPs.     

The preventive effect of Bacillus subtilus strain DB9011 against experimental infection with enterotoxcemic Escherichia coli in weaning piglets

Porcine edema disease (ED) is caused by Shiga toxin 2e‐producing Escherichia coli (STEC). Post‐weaned piglets often suffer from ED as a result of intestinal infection with STEC, which causes impaired growth performance and high mortality. Antimicrobial therapy is a curative treatment for piglets infected with STEC, but the emergence of antimicrobial‐resistant STEC has become a serious problem for Japanese pig farmers. Therefore, an alternative strategy other than antimicrobial therapy is needed for the prevention or treatment of ED. In this study, we evaluated the effect of oral administration of Bacillus subtilis DB9011 (DB9011) to prevent the experimental infection of STEC in weaning piglets. Eight 21‐day‐old piglets were divided into two groups: STEC challenge with the basal diet, and STEC challenge with DB9011 supplemented diet. The challenge was carried out when the animals were 25, 26 and 27 days old using STEC contained in capsules resistant against gastric digestion. All pigs were euthanized at 36 days of age. DB9011 improved the symptoms of ED and decreased the number of STEC in the ileal digesta and feces. Accordingly, oral administration of DB9011 in weaned piglets prevents ED through the suppression of the growth of STEC in the ileum.     

Shiga toxin 2eB‐transgenic lettuce vaccine is effective in protecting weaned piglets from edema disease caused by Shiga toxin‐producing Escherichia coli infection

Porcine edema disease (ED) is a toxemia that is caused by enteric infection with Shiga toxin 2e (Stx2e)‐producing Escherichia coli (STEC) and is associated with high mortality. Since ED occurs most frequently during the weaning period, preweaning vaccination of newborn piglets is required. We developed stx2eB‐transgenic lettuce as an oral vaccine candidate against ED and examined its protective efficacy using a piglet STEC infection model. Two serially developed Stx2eB‐lettuce strains, 2BN containing ingredient Stx2eB constituting a concentration level of 0.53 mg Stx2eB/g of powdered lettuce dry weight (DW) and 2BH containing ingredient Stx2eB constituting a concentration level of 2.3 mg of Stx2eB/g of powdered lettuce DW, were evaluated in three sequential experiments. Taken the results together, oral administration of Stx2eB‐lettuce vaccine was suggested to relieve the pathogenic symptoms of ED in piglets challenged with virulent STEC strain. Our data suggested that Stx2eB‐lettuce is a promising first oral vaccine candidate against ED.     

Nosological study of Borna disease virus infection in race horses.

To investigate the prevalence of diseases in the Borna disease virus (BDV) antibody positive race horses, we undertook seroepidemiological studies of BDV infection on 125 culled race horses in Hokkaido, Japan. The serological study disclosed the presence of antibodies only to BDV-p40 or -p24 in 19.2% (24/125) and 3.2% (4/125) of culled horses, respectively. Antibodies to both BDV-p40 and -p24 were found in 24.0% (30/125) of these horses. Of particular note was the finding that locomotorium disorders were detectable at a significantly higher rate in BDV antibody positive horses than that in the seronegative horses. These results imply that BDV infection may possibly contribute to an increase in the incidence rate of locomotorium disorders in race horses.     

Long-term changes in the chemical properties of Japanese cedar (Cryptomeria japonica) forest soils under high precipitation in southwest Japan

There is insufficient direct evidence of long-term changes in the chemical properties of forest soils to provide information for forestry management plans for sustainable site productivity. To understand changes in the chemical properties of forest soils in a specific high-precipitation climate in Japan, we re-investigated, in 1997, soil pits within a Japanese cedar (Cryptomeria japonica) forest in the Yanase region of Kochi Prefecture which had previously been surveyed in 1976. Comparison of the 1976 and 1997 results revealed significantly lower pH of surface and subsurface soils, and fewer exchangeable cations over time. In contrast, subsoil pH, concentrations of total carbon and nitrogen, and cation-exchange capacity (CEC) had not changed over the 21-year period. Estimation of proton and cation budgets in the forest ecosystem over 21 years, using data for the amounts in soil, biomass accumulation, and input through precipitation, revealed that biomass accumulation did not explain the cation depletion in soils. This suggests that cations were leached from the ecosystem. In contrast with previous reports, which showed Ca accumulation in Japanese cedar forest soils, our results indicated that high precipitation of more than 4,000 mm combined with acid deposition resulted in soil acidification and leaching of cations from soils, following the decrease in base saturation.     

Size-dependent impacts of invasive alien crayfish on a littoral marsh community

The invasive signal crayfish (Pacifastacus leniusculus) may undergo ontogenetic diet shifts with increasing size, but the consequences of crayfish size-structure changes for aquatic food webs are little known. We evaluated size-dependent impacts of signal crayfish on a littoral marsh food web using in-situ enclosures in a northern Japanese marsh. During the 60-day experiment, large crayfish (<30 mm orbital carapace length) rapidly eliminated submerged macrophytes through mechanical destruction while the comparable impacts of small crayfish (>30 mm) appeared after more time had passed. Benthic algal biomass was reduced in the presence of large crayfish but was affected little by small crayfish, suggesting that large crayfish were significant bioturbators. On the other hand, total invertebrate biomass was reduced while total invertebrate density was enhanced in the presence of both crayfish size classes. Potential crayfish impacts on invertebrates are: (1) predation on large vulnerable taxa such as Trichoptera and predatory invertebrates, (2) mechanical destruction of macrophytes, thereby reducing microhabitats for invertebrates, (3) heightened emigration of invertebrates in response to bioturbation and/or (4) predation or competition release on small invertebrates as a result of crayfish reducing their predators/competitors. Invertebrate taxa richness was nearly halved in the presence of large crayfish, probably reflecting reductions of rare taxa. Overall, functional roles of signal crayfish as omnivores and ecosystem engineers remained similar during the ontogenetic stage but the magnitude and rate of crayfish impacts intensified with increasing crayfish size. Thus, removal of large invasive crayfish alone may reduce detrimental impacts on aquatic biodiversity.     

Field study of bovine coronavirus vaccine enriched with hemagglutinating antigen for winter dysentery in dairy cows

A field study of a vaccine; prepared by solubilizing cells infected with bovine coronavirus, Triton X-100, and mixing with an oil adjuvant, was performed at 9 farms over 4 prefectures. The cattle tested were Holstein dairy cows aged 2 to 10 years. A vaccination group consisted of 157 animals (including 132 pregnant cows) and a non-vaccinated control group consisted of 50 animals. The cows received 2 intramuscular injections of vaccine (2 mL) at 3-week intervals. Vaccinated cows did not develop abnormalities, such as a decrease in milk production volume, and all pregnant animals calved normally. The geometric mean of the hemagglutination inhibition antibody titer was 34.2 before vaccination in test cows. The titer had increased to 105.6, 3 weeks after the 1st injection and peaked at 755.6, 1 month after the 2nd injection. A high antibody titer persisted at 396.0; 241.0; and 201.5, at 3, 6, and 9 months after the 2nd injection, respectively.

This confirms the safety and high antibody-response induced by this prototype vaccine. Therefore, this vaccine may be useful for the prevention of winter dysentery caused by bovine coronavirus infection.

     

Microscopic structure of the large intestinal mucosa in piglets during an antibiotic-associated diarrhea

Antibiotic-associated diarrhea (AAD) is caused by the treatments of broad-spectrum antimicrobials that seriously affect the activity and composition of the large intestinal microflora. The pathogenic bacteria or low concentration of short-chain fatty acids (SCFAs) has been repeatedly discussed in relation to AAD. Recently, we reported the detection of a large amount of succinate and lactate in the diarrheal feces in AAD-induced piglets. In this study, we investigated histologically the large intestinal mucosa in AAD-induced piglets, in which succinate and lactate were accumulated. AAD was induced in the piglets by an oral dose of polymyxin B sulfate (PL) or by an intra-muscular injection of enrofloxacin (ERFX). When the piglets were defecating diarrheal feces with a high concentration of succinate and/or lactate, the large intestine was removed and separated into four segments (cecum, gyri centripetales, gyri centrifugales, and rectum). Healthy piglets were used as the control. In the AAD-induced piglets, the lamina propria was edematous in the gyri centripetales. Piglets treated with ERFX were also edematous in gyri centrifugales. These edematous lamina propria contained larger amounts of inflammatory cells than observed in control tissues. ERFX-treated piglets had a more shallow crypt than PL-treated and control piglets. The mucosal tissue of the large intestine was more seriously damaged in the ERFX- than in the PL-treated piglets, which might have been caused by the high succinate and low SCFAs concentration in the digesta.     

In vitro shoot development ofEucalyptus citriodora on Rockwool in the film culture vessel under CO2 enrichment

An efficient system for growingin vitro plantlets ofEucalyptus citriodora Hook was developed. In the conventional closed system of culture with 2% sugar-containing gellan gum Murashige and Skoog (MS) medium supplemented with 0.02 mg/l indole-3-isobutyric acid, a serious defoliation of shoots was observed after three weeks. In contrast, plantlets grown on the sugar-free MS medium in the aerated bottle under 3,000 ppm CO₂ enriched condition did not show any defoliation. A marked enhanced growth of plantlets and no defoliation were observed on rockwool with the sugar-free liquid MS medium in the “Culture Pack”, made of fluorocarbon polymer film, under CO₂ enriched condition. CO₂ enrichment for this sugar-free “Culture Pack”-Rockwool system was also found to contribute to an improved growth of the plants in acclimatization. A part of this paper was presented at the 106th Annual Meeting of the Japanese Forestry Society (1995).