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Helicobacter pylori (H. pylori), first identified by Marshall and Warren, is a gram-negative bacterium found on the luminal surface of the gastric epithelium...

Background/Purpose Whether absorption of verotoxin (VT) 2 from the intestine in mice is inhibited by administration bovine immune colostral antibody against VT2 was investigated. Methods Three-week-old mice were administered VT2 solution at 477.8 ng/mL or 955.6 ng/mL, and bovine immune colostral antibody against VT2 was then administered three times. Whey without antibody against VT2 was administered to control mice. Serum levels of VT2 were measured by fluorescence enzyme immunoassay. Results Serum levels of VT2 in mice administered VT2 solution at 477.8 ng/mL and bovine immune colostral antibody against VT2 scarcely changed. By contrast, serum levels of VT2 in control mice increased and peaked 12 hours after administration. Peak values were 15.4 ± 5.04 ng/mL. Furthermore, serum levels of VT2 at 12 hours and 16 hours in control mice were significantly higher than in mice administered bovine colostral antibody against VT2. Serum levels of VT2 in mice administered antibody at 955.6 ng/mL showed no significant differences between repeated administration of bovine immune colostral antibody and controls. Conclusion These results suggest that absorption of VT2 from the intestine was inhibited by repeated administration of bovine immune colostral antibody against VT2 at early stages of Escherichia coli O157:H7 infection, whereas VT2 in the intestine remained at low levels.

Background. The prevalence of allergies is steadily increasing worldwide; however, the pathogenesis is still unclear. We hypothesized that Mycobacterium avium subsp. paratuberculosis (MAP) may contribute to allergy development. This organism can be present in dairy foods, it can elicit an immunomodulatory switch from a Th1 to a Th2 response, and it has been speculated that it is linked to several human autoimmune diseases. To determine the contribution, sera from 99 individuals with various atopic disorders and 45 healthy nonallergic controls were assessed for total IgE levels and successively for MAP-specific IgE by ELISA. Results. The mean total serum IgE level in allergic patients was 256±235 IU/mL, and in the healthy controls it was 62±44 IU/mL (AUC = 0.88; p<0.0001). Among the patient groups, 50 of the 99 subjects had increased IgE total level ≥ 150 IU/mL, while 49 subjects had IgE ≤ 150 IU/mL (mean level: 407±256 IU/mL versus 106±16 IU/mL; p<0.0001). Additionally, 6 out of 50 subjects (12%) with IgE ≥ 150 IU/mL and none (0%) with IgE ≤ 150 IU/mL were positive for specific MAP IgE (AUC = 0.63; p=0.03). Conclusion. The present study revealed that MAP has the ability to induce specific IgE and might contribute to the induction of allergic inflammation in genetically predisposed individuals.

The superoxide dismutase (SOD) gene clusters, sodB and sodC, and their adjacent genetic loci from a urease-positive thermophilic Campylobacter (UPTC) CF89-12 strain were analyzed molecularly, and compared with those of thermophilic campylobacters. The UPTC CF89-12 strain carried sodB [structural gene 654 base pairs (bp)] and sodC (540 bp) genes, as did the Campylobacter lari RM2100 reference strain. However, the other three thermophilic Campylobacter jejuni, C. coli and C. upsaliensis reference strains carried only a single sodB gene, and no sodC. Although sodB and sodC in the UPTC strain shared relatively high nucleotide sequence similarities (92.9 % and 91.7 %, respectively) with the corresponding genes in the C. lari RM2100 strain, the sodB gene in the UPTC CF89-12 and C. lari RM2100 strains shared relatively low nucleotide sequence similarities with those in C. jejuni NCTC11168 (80.8 % and 81.7 %), C. coli RM2228 (82.0 % and 83.1 %) and C. upsaliensis RM3195 (75.9 % and 77.0 %), respectively. All PCR amplifications of sodB and sodC gene segments with 28 C. lari isolates, including 14 UPTC isolates, gave positive results. C. lari organisms were shown to carry both the sodB and sodC genes with extremely high frequency. More high-SOD activity was seen with the C. lari isolates (n = 9), including UPTC, than was seen with the other three thermophilic Campylobacter and Helicobacter pylori organisms.

Synthesis of α2-macoglobulin (α2M) by 3-week-old juvenile rats was compared to that of mature 7- and 11-week-old rats. Serum concentrations of α2M, interleukin (IL)-6- and cytokine-induced neutrophil chemoattractant (CINC)-1 were measured by enzyme-linked immunosorbent assay. The area under the concentration vs. time curve (AUC) for α2M was significantly different among the three groups. The synthesis of α2M increased in an age-dependent manner. No significant difference was observed for the AUC of IL-6, but that of CINC-1 in 3-week-old rats was significantly lower than that in 7- or 11-week-old rats. These results suggest that synthesis of α2M was increased in mature compared to juvenile rats, possibly due to differences in liver function. The maximum concentration of CINC-1 in 3-week-old rats was observed 6 h after turpentine oil injection. The serum concentrations of IL-6 and CINC-1 increased more quickly in juvenile rats than in mature rats after inflammatory stimulation.

The changes in quantity of Cry j1 in Cryptomeria japonica pollen were assessed during the course of spring in Japan. C. japonica pollen was collected from the same trees in Hiroshima and Yamanashi Prefectures, and Cry j1 was quantified by enzyme-linked immunosorbent assay. The quantities of Cry j1 were found to gradually decrease, and this trend was similar in both Hiroshima and Yamanashi Prefectures. In Yamanashi Prefecture, no significant differences in the quantities of Cry j1 between the upper and lower parts of the tree were observed in samples collected from March 1 to 14. Furthermore, the quantities of Cry j1 on March 1, 5, 10 and 14 were significantly lower than those on February 1. These results suggest that expression of C. japonica pollen levels in terms of particle numbers is not sufficient and that quantities of Cry j1 vary throughout the course of spring.

Purpose The efficacy of bovine immune colostral (colostral) antibodies against verotoxin (VT) 2, flagellum and somatic cells of Escherichia coli ( E. coli ) O157:H7 in mice was determined. Methods Three major immunoglobulin (Ig) classes were isolated from the colostral antibody against VT2 by affinity chromatography and were used for estimation. Mice inoculated with VT2 were administered each Ig class from the colostral antibody, colostral antibody (colostral whey containing antibody) or serum antibody against VT2 at 1 hour after VT2 inoculation. Results All control mice (20/20) died after administration of sterilized saline instead of the colostral antibody. The survival rate was 93.3% (14/15) after administration of S-IgA or IgM antibody, or colostral antibody. Survival rates for IgG antibody and serum antibody administration were 80% (12/15) and 60% (9/15), respectively. Serum concentrations of VT2, which was absorbed from the small intestine in mice after administration of VT2 and colostral antibody, were measured by fluorescence enzyme immunoassay (FEIA). Serum concentrations of VT2 after administration of colostral antibody were lower than those after administration of sterilized saline. Mice inoculated with VT2-producing E. coli 157:H7 were administered anti-flagellum or anti-somatic colostral antibodies. Survival rates for E. coli O157:H7-infected mice administered the anti-flagellum and anti-somatic colostral antibodies were 52.4% (11/21) and 22.2% (4/18), respectively. Furthermore, survival rates increased to 89.5% (17/19) with combined administration of anti-flagellum and anti-VT2 colostral antibodies. Conclusion These results suggest that colostral antibodies against VT2, flagellum and somatic cells are effective against E. coli O157:H7 infection.

Recombinant full-length urease gene cluster and seven 100% deletion recombinant variants of urease subunits genes, (ureG, ureH, ureA, ureB, ureC, ureE and ureF) were constructed in vitro from the Campylobacter sputorum biovar paraureolyticus LMG17591 strain and expressed in Escherichia coli JM109 cells. A urease-positive reaction (1.885 micromol/min/mg protein) in the log-phase cultured E. coli cells transformed with pGEM-T vector carrying the recombinant full-length urease genes cluster was detected. Among the seven 100% deletion recombinant variants, each of the ureG-, ureH(D)-, ureA-, ureB-, ureC-, ureE- and ureF-deletion variants showed no change in assay of the urease reaction, and similarly as in the E. coli cell lysate with pGEM-T vector only. Recombinant full-length urease gene cluster and 100% deletion recombinants of the ureE gene in the transformed and log-phase cultured E. coli cells from the C. sputorum showed positively accelerated urease activities when cultured in the medium containing NiCl2 (750 micromol/L), but no activity was accelerated in the C. sputorum cultured in NiCl2. In addition, thiourea (20 mmol/L) completely inhibited urease activities from all C. sputorum examined. The putative recombinant urease subunits A and C were immunologically identified by Western blot analysis with polyclonal anti-urease alpha (A) and beta (B), raised against Helicobacter pylori.

The aim of this study was to investigate the synthesis of α2-macroglobulin (α2M) in hepatopathic rats injected with turpentine oil to induce acute inflammation. Hepatopathy was induced by oral administration of acetaminophen at a dose of 1 g/kg daily for 2 weeks or a 25% solution of carbon tetrachloride (CCl4) at 2 ml/kg body weight three times per week for 7 weeks. Acute inflammation was induced by intramuscular injection of turpentine oil at a dose of 1.0 ml/kg body weight. Serum concentrations of α2M were measured by enzyme-linked immunosorbent assay. Aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase and total protein differed significantly between acetaminophen or CCl4-induced hepatopathic rats and acetaminophen control (AA-control) or CCl4 control (CC-control) rats. Furthermore, pathological examination confirmed hepatopathy in rat livers. Peak serum concentrations and area under the time-concentration curve for α2M showed significant differences between hepatopathic rats and AA-control or CC-control rats. Thus, serum concentrations of α2M did not increase when compared with nontreated rats.

We investigated the effects of interleukin-6 (IL-6) and cytokine-induced neutrophil chemoattractant-1 (CINC-1) on α(2)-macroglobulin (α2M) production in rats. IL-6-rich and CINC-1-rich fractions were separated from serum obtained from rats 12 h after injection with turpentine oil using gel-chromatography. Sexual dimorphism was observed in the peak levels of α2M after injection of IL-6-, CINC-1-, or a mixture of IL-6-and-CINC-1-rich fractions. No significant differences in α2M levels were observed in males after injection with IL-6- or CINC-1-rich fractions and those injected with normal serum obtained from healthy rats (control). In contrast, serum levels of α2M, 6 to 120 h after injection of a mixture of IL-6- and CINC-1-rich fractions were significantly higher than in control rats. These results suggest that IL-6 and CINC-1 contribute to α2M production in rats only when IL-6 and CINC-1 act synergistically.

Resistance to intestinal proteases and efficacy against verotoxin (VT) 2 of a colostral antibody administered orally via catheter were investigated in beagle dogs. Bovine colostral antibody remained in the small intestine for 2 hours, whereas little serum antibody remained at 1.5 hours after administration. The bovine colostral antibody was not inactivated by proteases in the small intestine. Furthermore, the antibody activity of S-IgA did not change until 2 hours after administration; however, the activity of IgG and IgM antibodies decreased by two-thirds and two-fifths at 2 hours after administration, respectively. Seven beagle dogs inoculated with Escherichia coli (E. coli) O157:H7 producing VT2 were administered bovine colostral antibody or bovine colostral whey without antibody. With administration of bovine colostral whey without antibody, the amount of VT2 in feces decreased gradually after administration and increased again at 5 days after inoculation, while bovine colostral antibody significantly reduced the amount of VT2 in feces the day after administration. In addition, 9 beagle dogs were administered bovine colostral antibody, bovine plasma antibody or saline. The amount of VT2 in feces also significantly reduced rapidly after administration of bovine colostral antibody than administration of bovine plasma antibody or saline.

ヘモグロビンに代わる潜血の仮の指標として糞便および尿中の炭酸脱水酵素アイソエンザイム-I(CA-I)濃度を酵素免疫測定法(ELISA)により評価した。健康な各種年齢の実験用ビーグル犬113頭(雄50頭,雌63頭)における糞便中のCA-I濃度は,4.3から16.7ng/g便(平均;7.0±2.9ng/g便)であった。3頭の健康なビーグル犬から採取した血液1ml中に含まれるCA-Iは,それぞれ1,047, 1,062および1,150μgであった。自己血(10ml)を胃内へ注入したイヌの糞便CA-I濃度は大変低かった。しかし,自己血(5ml)を上行結腸部へ注入したイヌの糞便CA-I濃度は大変高かった。糞便中のCA-Iの検出は大腸からの出血があるイヌを見分けるのに有用であろう。健康な55頭のビーグル犬から採取した尿を化学的検査で調べた結果,44頭が陰性であったが,これら尿中のCA-I濃度はELISAで1.8から12.6ng/ml(平均;6.9±5.4ng/ml)であった。また,尿の潜血検査の結果が陽性であった11頭のイヌのCA-I濃度は,ELISAで41.2から525.0ng/mlであった。CA-Iは赤血球の特異的な指標ではないが,Hbに対する抗体を用いた特異的な免疫学的検査キットが開発されるまで,CA-Iはイヌの糞便および尿の潜血の検出に用いられるであろう。, This study was undertaken to investigate whether the concentration of carbonic anhydorase isoenzyme I (CA-I) in canine faeces and urine is useful as a temporary marker of occult blood. Concentrations of CA-I were measured by enzyme-linked immunosorbent assay (ELISA). The reactivity of canine CA-I did not change for 12 h at room temperature. Faecal CA-I concentrations in 113 healthy beagle dogs (50 male and 63 female) of various ages ranged from 4.3 to 16.7 ng/g faeces (mean; 7.0 ± 2.9 ng/g faeces). One ml of blood from 3 healthy beagle dogs were found to contain 1,047, 1,062 and 1,150 μg CA-I. The faecal CA-I concentrations of dogs receiving intragastric infusion of autologous blood (10 ml) were very low. However, the faecal CA-I concentrations of dogs receiving infusion of autologous blood (5 ml) into the ascending colon were very high. Of 55 urinary samples collected from healthy beagle dogs by catheter, chemical tests for occult blood were negative in 44, but CA-I concentrations ranged from 1.8 to 12.6 ng/ml (mean; 6.9 ± 5.4 ng/ml) by ELISA. The CA-I concentrations of the other 11 samples, which tested positive for occult blood on chemical testing, ranged from 41.2 to 525.0 ng/ml by ELISA.

我々は,セロトニン受容体のような神経伝達物質受容体あるいはそのトランスポーター(例えばセロトニン・トランスポーター(5-HTT))の遺伝子多型とオランザピンのような非定型抗精神病薬との関係を調査した。その結果,5-HTTのs対立遺伝子をもつ患者ともたない患者との間で,統合失調症陰性症状に対するオランザピンの効果に,統計学的な有意差が見られた。一方,アルツハイマー病患者に認められる精神神経症状に関わる5HT2A受容体と神経機能に重要な役割を持つダウの関係を明らかにするための予備段階として,2つのダウ・アイソフォーム(T3RとT4R)を明確に識別できる特性の高いポリクローン抗体を作製した。これらの抗体を用いて5HT2A受容体とタウとの関係を明らかにするために免疫組織化学的解析等を進める予定である。, We investigated the relationships between the effects of atypical antipsychotics such as olanzapine and neurotransmitter receptor and transporter gene polymorphisms such as serotonin receptor and transporter (5-HTT). There was a significant difference in the effect of olanzapine on the schizophrenia negative syndrome among the patients with and without the s allele of 5-HTT. Two types of tau isoform are found in neurofibrillary tangles-a pathological hallmark of tauopathies. To study how and which tau isoforms contribute to neurobal degeneration, we have developed two novel conformation-sensitive antibodies. There was no cross-reactivity between T3R (tau isoform, three-repeat tau) and T4R (four-repeat tau), and T3R and T4R showed reduced binding to the thioflavin-positive β-structural form of their target.

異なる炎症刺激を与えて急性炎症を惹起させた9週齢のSprague-Dawley系雄性ラットにおいて,α_1-酸性糖蛋白(α_1・AG),インターロイキン(IL)1,6およびCytokine-induced neutrophil chemoattractant-1(CINC-1)の血清中濃度の推移を検討した。α_1・AGは,炎症刺激後48時間に最高値を示した。そのα_1・AG値は,テレピン油(0.4ml/head)投与例では投与前値の33.7倍で,インドメタシン(20mg/kg)投与例では投与前値の15.5倍であった。CINC-1は,テレピン油,インドメタシンおよび黄色ブドウ球菌投与後に増加し,投与12時間後に最高値を示した。しかし,IL-6は,テレピン油投与時のみに増加(最高値27.4倍)し,インドメタシンおよび黄色ブドウ球菌投与時には増加を示さなかった。テレピン油投与例でIL-1に変化は認められなかった。, Serum levels of α_1・AG, IL-1, IL-6 and CINC-1 were estimated in twenty-five 9-week-old male SD rats following various types of acute inflammatory stimulation. Serum levels of α_1・AG and IL-6, CINC-1 were measured by SRID and ELISA, respectively, using commercial kits. α_1・AG increased after inflammatory stimulation and peak levels were observed after 48h. Serum levels of α_1・AG also increased after inoculation with S. aureus, but were scarcely increased after inoculation with P. aeruginosa. However, only CINC-1 increased after injection of indomethacin (20mg/kg); IL-6 levels did not change. This phenomenon was also observed in rats inoculated with S. aureus. IL-6 increased only after injection of turpentine oil and did not change after injection of indomethacin or inoculation with S. aureus. IL-1 did not change after injection of turpentine oil. α_1・AG is useful as an inflammatory marker in rats

Mycobacterium avium subsp. paratuberculosis (MAP) is the established causative agent of Johne's disease in cattle and other ruminants, and it has also been speculated to be a putative etiological agent of several human autoimmune diseases. It is acknowledged that dairy products deriving from infected animals play a role (could be vehicles) in exposing humans to MAP. MAP could stimulate the human immune system by means of their complex antigen (in the case of lipids, multivalent antigens) and may modulate it, acting as adjuvant molecules such as Freund's complete adjuvant. The immune system might be abnormally stimulated by the constant presence of MAP antigens (for example, in the dairy products), and this might be particularly relevant in genetically predisposed individuals. However, there is limited understanding about the current human exposure to MAP. The present study analyzed the antibody recognition profile of MAP lipophilic antigens in a cohort of 126 healthy Japanese. We measured the serum levels of total immunoglobulin G (IgG) and subclasses targeting MAP surface antigens through ethanol vortex indirect enzyme-linked immunosorbent assay (EVELISA) by using serum absorbed with Mycobacterium phlei. Elevated IgG (especially IgG1 and IgG4) responses were observed in 14% of the sera. To assess the specificity of EVELISA, the same samples were analyzed by means of a commercially available Johnelisa II kit. It was noteworthy that a high degree of correlation was observed when comparing the two methodologies (rs=0.7, p0.0001). Moreover, in order to investigate the specificity of the binding, inhibition assay experiments were carried out also searching for antibodies against Bacillus Calmette-Guérin antigens, but no cross-reaction was observed. The result obtained represents the first evidence implying that the Japanese population is exposed to MAP, and additionally the existence of a foodborne chain of exposure that transmits MAP antigens to humans.

The half-lives of typical acute phase proteins in rats and beagle dogs during acute inflammation were investigated. Acute inflammation was induced by injection of turpentine oil in rats and administration of indomethacin in beagle dogs. Serum concentrations of α2-macroglobulin (α2M) and C-reactive protein (CRP) were measured by enzyme-linked immunosorbent assay and α1-acid glycoprotein (AAG) was measured by single radial immunodiffusion. Half-life was calculated as 0.693/elimination rate constant (K). The mean half-lives in the terminal elimination phase of α2M and AAG were 68.1 and 164.8 h, respectively. The half-life of AAG was significantly longer than that of α2M. Mean half-lives in the terminal elimination phase of CRP and AAG were 161.9 and 304.4 h, respectively. The half-life of AAG was significantly longer than that of CRP in beagle dogs. No significant differences in the half-life of AAG were observed between rats and beagle dogs. Furthermore, serum concentrations in the terminal elimination phase could be simulated with the K data acquired in this study.

The neutralization efficacy of bovine colostral antibody against verotoxin (VT) 1 and 2 was investigated. Cows were immunized with VT1 or VT2 fourteen times at 7-day intervals. A colostral antibody exhibiting high titers was obtained from immunized cows. Survival rates were evaluated in mice administered VT1 or VT2, and those infected with Escherichia coli (E. coli) O157:H7 producing VT1 or VT2. Survival rates after VT1 administration were 100% in the single-administration group, 90% in the repeat-administration group, and 78.6% in the control group. Survival rates after VT2 were 75.0% in the single-administration group, and 100% in the repeat-administration group. All mice in the control group died. Colostral antibody and fosfomycin (FOM) in the colostral antibody group and FOM and skim milk in the control group were administered three times per day for 5 days to mice infected with E. coli O157:H7 producing VT1 or VT2. Survival rates after inoculation with E. coli O157:H7 producing VT1 were 80.0% in the colostral antibody group, and 63.6% in the control group. Survival rates after inoculation with E. coli O157:H7 producing VT2 were 83.3% in the colostral antibody group, and 20.0% in the control group. The survival rate in mice without treatment following inoculation with E. coli O157:H7 producing VT2 was 88.2%. The survival rates in mice infected with E. coli O157:H7 strains producing VT1 or VT2 improved after administration of this colostral antibody, which exhibited neutralization efficacy against VT.

健康な3～72ヵ月齢の雌雄ビーグル犬における血清CRPおよびAAG濃度には雌雄差および月齢差が認められなかった。しかし,AAG値は個体間によるばらつきが大きく,個体ごとの濃度の違いを十分に考慮した上で急性炎症の指標として用いる必要があると考えられた。また,妊娠期間中のCRPおよびAAG濃度は,ともに妊娠中期に上昇することが確認され,妊娠期間中における両濃度の解釈にはこのことを考慮する必要があると考えられた。また,母体の血清AAG濃度は高値を示したが,胎仔血清AAG濃度は検出限界以下であったことから,イヌのAAGには胎盤移行性がないことが強く示唆された。, Serum C-reactive protein (CRP) and α_1-acid glycoprotein (AAG) levels in healthy beagle dogs of various ages and in pregnant beagle dogs were measured. Serum CRP levels were 1.5-16.0μg/ml (mean 7.9±3.4μg/ml) in male dogs, and 1.8-18.9μg/ml (mean 8.3±4.0μg/ml) in female dogs by ELISA. No significant sex-related differences were observed in the values. Further, there were no significant age-related differences either. Serum CRP levels increased during pregnancy. The levels of serum CRP in pregnant beagle dogs peaked at 70.2-90.4μg/ml (mean 77.5±7.1μg/ml) 30 or 45 days after ovulation, demonstrating two characteristic features of CRP level change in pregnant dogs. AAG levels ranged from 40 to 960μg/ml (mean 322±202μg/ml) in male dogs, and from 47 to 833μg/ml (mean 316±199μg/ml) in female dogs by singl radial immunodiffusion (SRID), without any significant sexor age-related variation. Serum AAG levels increased in all pregnant beagle dogs and peaked in the middle of gestation at 250-1,000μg/ml (mean 634±246μg/ml). In 7 of 8 dogs the AAG levels peaked about 45 days after ovulation. Despite a high value of 1,210-1,360μg/ml being observed for serum AAG levels in 3 pregnant beagles inoculated with Staphylococcus aureus, its levels in umbilical cord blood were below the detection limit of SRID (40μg/ml).

レジオネラ属菌の除去を目的として,塩素系薬剤に替わる天然抗菌剤の検索を行うため,グレープフルーツ種子抽出物(GSE)について,L. pneumophilaに対する抗菌効果を検討したところ,以下の成績が得られた。1. L. pneumophilaに対するGSEの抗菌活性試験を行ったところ,供試した25菌株は17～25mmの範囲に明瞭な阻止円の形成を示し,平均値は20.8mmであった。2. L. pneumophilaに対するGSEのMIC値を測定したところ,範囲は97.7mg/lから390.6mg/lを示し,MIC_は390.6mg/lであった。3. L. pneumophilaに対するGSEのMBC値を測定したところ,範囲は接触時間1分,10分,60分で12,500～50,000mg/l,1,563～6,250mg/l,781.3～3,125mg/lを示し,MBC_は1分,10分,60分で,それぞれ50,000mg/l,6,250mg/l,3,125mg/lであった。4. 40℃に保温したpH7.0とpH9.0のリン酸緩衝液およびpH7.0のリン酸緩衝液に生薬を添加した溶液中にGSE100mg/lおよび1,000mg/lを含む溶液を用いて標準株と浴槽水由来株に対するGSEの殺菌効果を検討したところ,いずれの条件でも菌接種後1分以内で99.99%が殺菌された。以上のことから,GSEはL. pneumophilaに対して抗菌活性や殺菌効果を示すことが明らかとなった。, We investigated the antibacterial effect of commercial grapefruit seed extract (GSE, Liquid), which has recently been attracting attention as a food additive, on Legionella pneumophila. The sensitivity test was performed using the disc method. All 25 strains tested were sensitive to GSE, and formed clear inhibition zones in its presence. The minimum inhibitory concentration (MIC) values were measured by the agar dilution method. The MIC of GSE in the strains was in the range of 97.7 mg/l to 390.6 mg/l, and the MIC_ was 390.6 mg/l. The minimum bactericidal concentration (MBC) values for the test strains were also determined. MBC_ was 50,000, 6,250, and 3,125 mg/l when the exposure time was 1, 10, and 60 minutes, respectively. These bactericidal test results demonstrate that GSE has a bactericidal effect on L. pneumophila.

In the present study, three canine blood samples from Japan, that were suspected to be ehrlichia positive were examined. After sequencing the 16S rRNA genes, each dog was found to be infected either with Ehrlichia canis (Kagoshima 1), Anaplasma platys (Okinawa 1), or Wolbachia sp. (Okinawa 2). Phylogenic analysis was performed on these sequences. The nearly entire 16S rRNA sequence of Kagoshima 1 was found to be most similar to the sequences from Oklahoma and Venezuela E. canis strains (1 base pair difference out of 1,387, 99.9% sequence identity). The 16S rRNA gene sequence of Okinawa 1 showed the closest DNA identity to the French strain of A. platys (1 base deletion out of 1,385 bp, 99.6% sequence identity). The 16S rRNA gene sequence of Okinawa 2 illustrated the closest DNA identity to that of a Wolbachia sp. from Dirofilaria immitis (98.9% sequence similarity). These data imply a low diversity within E. canis strains and within A. platys strains, including those strains reported in this study. This is also the first demonstration of Wolbachia DNA in dog blood, suggesting the involvement of Wolbachia sp. in canine febrile illnesses.

本実験の結果から,中和抗体を高力価に含有するウシの初乳乳清は,動物の腸管内でべ口毒素の活性を中和できることが確認された。今回は乳清の1回のみの経口投与を行ったが,腸管内へは異種動物由来抗体の反復投与が可能であり,胃液を中和する前処理を行った上で,乳清を複数回投与することによってより高い中和効果が期待できるものと考えられた。ヒトのO157感染症の治療に際して,高力価の中和抗体を含有するウシの初乳乳清を併用することにより充分量の抗菌剤を投与してO157を死滅させる積極的な治療法が可能になるものと期待される。, The colostral secretory antibody to verotoxin (VT1・VT2, VT2) of enterohemorrhagic Escherichia coli O157 (EHEC) was prepared in 3 dairy cattle by immunization with VT1・VT2 mixture or purified VT2. The whey containing colostral secretory antibody was isolated from colostrum by addition of the rennet. The verotoxin-neutralizing antibody titers of colostral whey were 1: 256-1: 512 to VT1 and 1: 16-1: 64 to VT2. All of adult wister rats, ddy mice, ICR mice and beagle dogs were not sensitive to verotoxin. Three weeks ages of ICR mice were sensitive to VT2 and beagle dogs pre-administrated of Fradiomycin sulfate (50mg/kg) orally for 3 days excreted VT2 into feaces after inoculation of EHEC (1×10^7 cells). These animals were used for experiments in neutralizing VT2 activity in the gastrointestinal track. The levels of VT2 was measured by microtiter method using VTEC-RPLA and shown by end points. Seven ICR mice (3 weeks ages) were all died after 2 days of oral administration of VT2 (approximately 125 ng/0.5ml). However, 5 ICR mice (3 weeks ages) that administrated colostral whey containing neutralizing antibody to VT2 after administration of VT2 (approximately 125 ng/0.5ml) survived. Three beagle dogs administrated Fradiomycin sulfate have diarrhea after 2 days of EHEC inoculation. The control beagle dog excreted VT2 of 1:16 (approximately 25ng/ml) into faeces during 6 days. A beagle dog, that excreted VT2 of 1:32 (approximately 50ng/ml) into faeces after 3 days of EHEC inoculation, was administered orally 100ml of colostral whey containing neutralizing antibody (1:64). Levels of VT2 in faeces was decreased markedly to 1:8 at next day (4 days of EHEC inoculation) and 1:4 at 2 days later (5 days of EHEC inoculation). These results showed that the bovine colostral secretory antibody to VT2 has neutralizing activity without digestion by proteinase in the animal gastrointestinal track.

レジオネラ症に関する疫学的研究の一環として,2001年に全国の表層土壌から分離固定したレジオネラ属菌62株についてAcanthamoeba内での増殖能について検討したところ,供試菌株はすべてルAcanthamoeba内で増殖し,病原性を有するものと考えられた。また,増殖に要する最小菌数は10^3～10^8CFU/mlで,菌株によって差異が認められた。以上のように,土壌由来株はいずれも病原性であると考えられた。, As part of an epidemiological study of legionellosis, we investigated the growth within Acanthamoeba sp. Of 62 strains of Leginella spp. Isolated from surface soils nationwide in 2001. All strains tested grew in Acanthamoeba sp., suggesting that the strains were pathogenic. The minimum bacterial number required for the growth in the amoeba was 10^3-10^8 CFU/ml and there were differences between the strains. The above findings suggested that all soil-deriver strains were pathogenic.

Serum alpha 1-acid glycoprotein (AAG) levels were measured in healthy beagles of various ages (66 male and 74 female) by turbidimetric immunoassay (TIA), and then separately--in pregnant beagles--by single radial immunodiffusion (SRID). The first experiment revealed that serum AAG levels ranged from 40 to 960 microg/ml (mean of 322 +/- 202 microg/ml) in male dogs, and from 47 to 833 microg/ml--in female dogs (mean of 316 +/- 199 microg/ml), without any significant sex- or age-related variation. The second experiment, however, revealed that serum AAG levels increased in all pregnant beagles and peaked in the middle of gestation at 250-1,000 microg/ml (mean of 634 +/- 246 microg/ml). In 7 of 8 dogs the AAG levels peaked about 45 days after ovulation. Despite a high value of 1,210-1,360 microg/ml being observed for serum AAG levels in 3 pregnant beagles inoculated with Staphylococcus aureus, its levels in umbilical cord blood were below the detection limit of SRID (40 microg/ml).

The aims of this study were to investigate transplacental transport of alpha 2-macroglobulin (alpha 2M) in rats in rats and to examine the degree of alpha 2M induction in maternal and neonatal rats with acute inflammation. Serum was collected from healthy pregnant CD (IGS) rats, neonates of the pregnant rats and their cord blood. Additional serum samples were obtained from pregnant rats inoculated with an inflammatory agent, turpentine oil, their neonates and cord blood, and neonates inoculated with turpentine oil. The serum levels of alpha 2M were measured by means of an enzyme-linked immunosorbent assay. The average serum levels of alpha 2M in healthy neonates and cord blood were about 380 micrograms/ml. Serum a2M level in neonates inoculated with turpentine oil averaged about 580 micrograms/ml. Serum alpha 2M levels in maternal rats inoculated with turpentine oil, neonates from those rats and their cord blood were elevated, the values being 2,000 micrograms/ml or higher. It was demonstrated that induction of alpha 2M in neonatal rats was lower than in maternal rats when inoculated with turpentine oil. These results suggest that alpha 2M is transplacentally transported from maternal rats to fetal ones.

Summary An enzyme-linked immunosorbent assay (ELISA) for rat α2-macroglobulin (α2M) was developed, and the concentrations of α2M in sera in normal Crj:CD and Crj:WI rats as well as in turpentine oil injected Crj:WI rats were determined. Anti-rat α2M serum was prepared by immunization of rabbits with the α2M fraction separated from rat acute phase serum using Sephacryl S-300. The specificity of the rabbit anti-rat α2M serum was confirmed by immunoelectrophoresis and Western blotting. The α2M concentrations in 180 normal Crj:CD (90 males and 90 females) and 180 Crj:WI (90 males and 90 females) rats were 14.5–94.9 μg/ml (mean 36.8 ± 13.3) and 13.6–65.8 μg/ml (mean 34.9 ± 12.4), respectively. There were no significant differences between male and female Crj:CD and Crj:WI rats, respectively (p

To elucidate the source and route of VTEC infection, we performed pulse field gel electrophoresis (PFGE) an 50 isolates from human diarrhea typed as serotypes O157, O111, and O26, which were very frequently isolated from patients with VTEC infection between 1986 and 1997, and 32 isolates from dairy cattle, a total of 82 isolates. The isolates were genetically analyzed based on the electrophoresis patterns of DNA, and a phylogenetic tree was prepared. The isolates were classified based on similarity > or = 89. The following results of the molecular epidemiological investigation were obtained. 1) Based on the electrophoresis patterns of DNA obtained by PFGE, 34 of the 49 O157 isolates (69.4%) were divided into groups 1-9, 15 of the 18 O111 isolates (83.3%) were divided into groups 1-3, and 12 of the 15 O26 isolates (80%) were divided into groups 1-3. Of the grouped isolates, group 8 of O157, groups 2 and 3 of O111, and group 3 of O26 included isolates from human diarrhea and dairy cattle, but the other groups included isolates from only one of the two sources. 2) With regard to regional investigation, groups 6 and 9 of O157 included human diarrhea-derived isolates from Yokohama and Ehime, and group 8 included a human diarrhea-derived isolate from Yokohama and a dairy cattle-derived isolate from Tokushima. Group 3 of O111 included a human diarrhea-derived isolate from Ehime and a dairy cattle-derived isolate from Hokkaido. Group 3 of O26 included human diarrhea-derived isolate from Ehime and dairy cattle-derived isolate from Sagamihara and Hokkaido. Since the above findings showed that although the frequency was low, isolates from human diarrhea and dairy cattle were included in the same groups, it was demonstrated that dairy cattle are closely related to the human infectious disease of the intestinal tract as a source of infection. However, classification using the PFGE method is difficult due to diversity of the electrophoresis pattern of DNA. It is necessary to investigate the classification by a combination of the PFGE method with phage typing, ribotyping, and RAPD-PCR, and to investigate more numbers of patient-derived and animal-derived isolates.

An arsenate susceptibility test was performed with transformed and cultured Escherichia coli DH5α cells, which carried recombinant DNA of full-length arsenic (ars) operon, namely a putative membrane permease, ArsP; a transcriptional repressor, ArsR; an arsenate reductase, ArsC; and an arsenical-resistance membrane transporter, Acr3, from the Japanese urease-positive thermophilic Campylobacter lari (UPTC) CF89-12. The E. coli DH5α transformant showed reduced susceptibility to arsenate (~1536 μg/mL), compared to the control. Thus, these ars four-genes from the UPTC CF89-12 strain cells could confer a reduced susceptibility to arsenate in the transformed and E. coli DH5α cells. E. coli transformants with truncated ars operons, acr3 (acr3) and arsC-acr3 (∆arsC-acr3), of the ars operon, showed an MIC value of 384 μg/mL (~384 μg/mL), similar to the E. coli cells which carried the pGEM-T vector (control). Reverse transcription PCR confirmed in vivo transcription of recombinant full-length ars operon and deletion variants (∆acr3 and ∆arsC-acr3) in the transformed E. coli cells.

The application of the immunoperoxidase (IP) plaque staining procedure (IP test) to the diagnosis of canine coronavirus (CCV) infection was investigated. The IP test did not react with sera from either 15 specific pathogen-free (SPF) dogs or 7 SPF dogs immunized with a multivalent vaccine, including canine parvovirus type 2, canine distemper virus, canine adenovirus type 2, and canine parainfluenza virus. To compare the IP test with the neutralizing test (NT), sera from 240 healthy dogs and from 3 experimentally CCV-infected dogs were examined. All 60 sera positive for NT antibody were positive for IP antibody, and all 180 sera negative for NT antibody were negative for IP antibody in the healthy dogs. The IP titres showed similar changes with time after CCV inoculation to those of the NT titres in the experimentally infected dogs. These findings indicate that the IP test specifically detected anti-CCV antibodies. When the IP test and NT were compared in dogs with diarrhoeic signs. 2.1% of 48 sera and 20.3% of 74 sera, which were all negative for NT antibody, were positive for IP antibody in the dogs of under one year of age and at least one year of age, respectively. The difference between the IP and NT titres (log10 [reciprocal of IP titre] log10 [reciprocal of NT titre]) for the diarrhoeic dogs of under one year of age (2.350 +/- 0.931) was significantly larger than that for the healthy dogs (0.982 +/- 0.447) (p

The concentrations of C-reactive protein (CRP) and α1-acid glycoprotein (AAG) were evaluated in 1-, 3- and 18-month-old dogs (four of each age) that had been inoculated with turpentine oil. The CRP and AAG in 3-month-old and younger dogs subjected to surgery or inoculated with either Staphylococcus aureus or a viral vaccine were also evaluated. The average CRP concentration in the sera peaked 2 days after inoculation of turpentine oil. The peak CRP concentrations in 3- and 18-month-old dogs were significantly (p

The use of immunoperoxidase plaque staining (IP) in the antibody test for canine distemper virus (CDV) and its diagnostic value were investigated. Antibody titres by the IP test and neutralizing test (NT) against CDV were compared in 114 specific pathogen-free (SPF) dogs, including 84 CDV-vaccinated and 30 unimmunized dogs. All of the unimmunized dogs, which had negative NT titres, also showed negative IP titres, and all of the CDV-vaccinated dogs, which had positive NT titres, showed positive IP titres. Furthermore, the IP titres showed similar changes with time after vaccination to the NT titres in five CDV-vaccinated SPF dogs. These findings indicate that the IP test has similar specificity to the NT.

To establish the diagnostic significance of canine C-reactive protein (CRP) in gastrointestinal disorders, the serum canine CRP concentration was measured in dogs with experimentally-induced acute gastric mucosal injury. Gastric injury was induced in one male and one female beagle by a single dose oral administration of acetylsalicylic acid (200 mg/kg body weight) or indomethacin (60 mg/kg body weight), or sodium chloride (1000 mg/kg body weight). CRP was measured prior to dose, and 1, 3, 7, and 14 days after the administration of the drugs, together with the total leucocyte counts and serum iron. Changes in the serum CRP in dogs with gastric injury were similar for the three test compounds, and reflected by the endoscopic findings. CRP values increased from 87 to 390 mg/l within 1 to 3 days after the compound administration but returned nearly to the predose levels within 14 days. Endoscopy revealed haemorrhagic erosion of the gastric mucosa in all dogs one day after dosing, with no evidence of the erosions observed after 7 days in many of the dogs. Changes of the total leucocyte and serum iron also occurred following gastric injury, but these changes were not as marked as those observed for CRP. The results of this study suggest that serum CRP level may be a useful indicator of a gastrointestinal mucosal injury in dogs.

The cahge of serum levesl of α_2-macrogolbulin (α2M) after administration of fraction corresponded to interleukin (IL)-6 or cytokine-induced neutrophil chemoattractant-1 (CINC-1) in rats were investigated. The serum levels of α_2M increased franction of corresponded of IL-6 or CINC-1 and peak levels 260.7 μg/ml at 48 h and 357.5 μ/ml at 24 h after administration, respectively. These results suggested that IL- 6 and CINC-1 were presumed to be contributed production of α2M.

In this paper, the method of monitoring of development of crack of structural member caused by dynamical load by angular velocity sensors using vibratory gyroscope is proposed. The one story structure having the test column with the artificial crack was oscillated at the base, assuming ground motion. Then, the test column took the repeated bending moment and the crack at the tip of artificial one was devoloped. The two angular velocity sensors using vibratory gyroscope were installed at the same distance from the center of crack. By the measurement of angular velocities, the bending curvature of material were able to be measured. Then, it is possible to measure the development of crack in material undertaking dynamic load, because a development of crack gives the change of moment curvature dramatically. Through the experiment, it was proven that the proposed method is effective to find the development of crack in material taking dynamical load.

The concentrations of C-reactive protein (CRP) in serum from normal crab-eating monkeys (Macaca irus) were measured by means of a monkey-specific turbidimetric immunoassay (TIA), and the changes in the serum CRP concentrations in crab-eating monkeys inoculated with Bordetella bronchiseptica R-5 and measles virus (Ichinose or NK 3 strain) were also examined. The CRP concentrations in sera from 54 normal crab-eating monkeys ranged from 0 to 8.3 microg/ml (mean 2.2 +/- 1.9). No significant difference was found in the CRP concentrations between males and females (p > 0.05). The concentrations of CRP in the sera from four crab-eating monkeys inoculated intrabronchially with 10(9) live B. bronchiseptica increased gradually to a peak at 2 days after inoculation. The peak concentrations of CRP were from 102.4 to 313.2 microg/ml, 54-96 times the preinoculative values of 1.9-5.6 microg/ml. When the same four crab-eating monkeys were inoculated intrabronchially with measles virus 34 days after inoculation of B. bronchiseptica, the serum CRP concentrations did not increase. Monitoring of CRP is useful for assessing monkeys with acute B. bronchiseptica infection and will probably be of value in the diagnosis of other bacterial infections.

A machine and structure reduce their own strength by the cumulative damage caused by fatigue generated by dynamic loads. If the signals caused by the development of crack in materials are able to be detected, the condition of health of a machine and structure will be monitored to prevent the sudden fracture of them. It was reported by authors that the signals of that can be detected by the wavelet transform of the observed signals such as displacement, velocity and acceleration etc, because the wavelet transform can detect the discontinuity in signals. However, the wavelet transform require the computed time for its transform and then it cannot be used for on-line monitoring. Then, it has been strongly desired to detect the discontinuity in signal of acceleration and then, the jerk sensor composed by a vibratory gyroscope and a cantilever has been developed for this purpose. The proposed sensor is manufactured and used for the experiment, which prove the availabilty of this proposed sensor.

The aims of this study were to evaluate the imhibition of colonization and the neutralization of verotoxin (VT) of enterohemorrhagic Escherichia coli O157 colostral antibodies in beagle dogs. Cows were immunized with bacterial cells (O157) purified VT2 14 times at 7-day intervals. Colostral antibodies were obtained from immunized cows after delivery. The titer of bovine colostral antibody to VT was high. But, the titer of antibody to flagellum was very low. IgA antibody titer to VT was higher than IgG or IgM antibody titer. Beagle dogs inoculated with O157 producing VT2 were administered colostral antibody, serum antibody or saline. Furthremore, colostral antibody and colostrum whey obtained from non-immunized cow were administrated. The amount of VT2 in feces was reduced immediately by administration of colostral antibody than plasma antibody, saline or colostrum whey. The residual time in small intestine of bovine colostral antibody and plasma antibody obtained from rabitts immunized VT2 was compared. The residual time of bovine colostral antibody was longer than rabbit serum antibody. This result suggested that bovine colostral antibody resisted to protease in digestive organ.

Monkey-specific C-reactive protein (CRP) assay methods (enzyme-linked immunosorbent assay (ELISA) and turbidimetric immunoassay (TIA)) were developed. The anti-monkey CRP serum was prepared by immunization of rabbits with the immune complex formed between the acute-phase serum from turpentine oil-inoculated monkeys and goat anti-human CRP serum. The specificity of the rabbit anti-monkey CRP serum was confirmed by immunoelectrophoresis and Western blotting. The purity of monkey CRP prepared by chromatography procedures was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The serum CRP levels in nine normal monkeys, as measured by sandwich ELISA were ranged from 0.26 to 1.42 microg/ml (mean 0.71+/-0.37). The CRP levels in five acute-phase sera of turpentine oil-inoculated monkeys were 248-451 microg/ml (mean 371.2+/-73.8). This monkey-specific CRP assay method was found more sensitive than the human-specific CRP assay method in detecting monkey CRP by TIA.

Faecal haemoglobin (Hb) concentrations in apparently healthy experimental Beagle dogs and in dogs of various breeds kept in private households or at breeders were measured by reversed passive latex agglutination (RPLA) and enzyme-linked immunosorbent assay (ELISA) in an effort to define the physiological concentrations of faecal Hb in the dog. In 88% (53) of 60 experimental Beagle dogs (30 males and 30 females), the RPLA titres were 1:2 and 1:8 and the faecal Hb concentrations ranged from 40.0 to 431.5 (mean 184.1±92.6) μg/g faeces by ELISA. No significant difference was found in Hb levels or RPLA titres between males and females. Seven dogs (12%) had significantly greater RPLA titres and Hb concentrations by ELISA than the remaining dogs. In 84% (45) of the 53 dogs kept in private households or at breeders, the RPLA titres were >1:1 to 1:8 and the faecal Hb concentrations ranged from 7.1 to 456.7 (mean 137.5±128.7) μg/g faeces in ELISA. Eight of these dogs (15.1% of 53 dogs) had significantly greater RPLA titres and Hb concentrations by ELISA than the remaining dogs. There were no significant differences between the Beagles and dogs kept in private households or at breeders. In conclusion, in 98 (86.7% of 113) dogs the physiological concentrations of RPLA titres were >1:1 to 1:8 and the faecal Hb concentrations were 143.5–185.1 μg/g (95% confidence level). Approximately 13.3% of apparently healthy dogs had higher faecal Hb concentrations, suggesting the presence of subclinical haemorrhages. Four dogs suffering from colorectal cancer also had high faecal Hb concentrations.

The sound field coupled with inner and outer space separated at an enclosure has been analyzed by using the boundary element method and reported. It has been available that the most of sound energy more than 80% is flooded from the opening of the enclosure. Then, it is desired that the sound energy shall be decreased while it pass through the neck of opening of enclosure and the passive sound control system, which is consisted of the cave in the neck and the damping attached to the inside wall, is proposed for this object. The sound passing the neck of opening of enclosure is introduced to this sound control system and the most of sound is dissipated by damping material attached to the inside of the system. The effectiveness of this system is proven by computer simulation and experiment.

This study was undertaken to investigate whether the level of C-reactive protein (CRP) in the serum of dogs undergoes physiological variation, using 10 normal Beagle dogs (5 males and 5 females), 1-2 years old, maintained in a healthy condition in a controlled environment. The CRP concentration in the sera collected seven times each day at intervals of approximately 3 h ranged from 0.8 to 16.4 micrograms/ml (mean 5.06 +/- 3.60) in one experiment and from 0.8 to 14.0 micrograms/ml (mean 4.50 +/- 2.80) in a second experiment. On examining the 24-h variations in the concentration of CRP in serum, neither consistent changes nor a definite pattern of circadian rhythm was detected. During 28 days observation, only very slight changes, which seemed attributable to analytical errors, were seen in any of the dogs, except one. The concentration of CRP in the serum during the 28 days ranged from 0.8 to 22.6 micrograms/ml (mean 3.65 +/- 1.40). The concentrations underwent no significant variations in individual dogs, but significant differences were found between the dogs (p < 0.01).