Chemotactic activity of hemocytes derived from a brackish-water clam, Corbicula japonica, to Vibrio parahaemolyticus and Escherichia coli strains.

Hemocytes from adult and juvenile specimens of a brackish-water clam, Corbicula japonica, were attracted chemotactically to live cells of Vibrio parahaemolyticus and Escherichia coli strains in a balanced salt solution, which was enhanced significantly in the presence of respective C. japonica plasma. Chemotactic attractions of adult's and juvenile's hemocytes were seen also in artificial seawater at a similar level to those in the balanced salt solution. Chemotactic attractions of juvenile's hemocytes to these strains were lower in level than those of adult's hemocytes. C. japonica plasma seems to facilitate for C. japonica hemocytes to recognize these organisms.     

In vitro attachment of Vibrio parahaemolyticus to hemocytes of two gastropod molluscs.

Vibrio parahaemolyticus D-3 was observed to attach to hemocytes of a marine gastropod mollusc, Nerita albicilla, regardless of the presence of N. albicilla serum. The organism attached to hemocytes of an estuarine gastropod, Clithon retropictus, in the presence of C. retropictus serum while the attachment to the hemocytes was decreased significantly in the absence of the serum. These evidences suggest that N. albicilla hemocytes would facilitate the clearance of V. parahaemolyticus from the alimentary tract of the mollusc and that C. retropictus hemocytes would protect C. retropictus against the invasion of V. parahaemolyticus to hemocoel of the mollusc.     

Migratory responses of hemocytes to Vibrio parahaemolyticus in the alimentary tract of an estuarine neritid gastropod, Clithon retropictus.

Migratory responses of hemocytes to Vibrio parahaemolyticus strain D3 in the alimentary tracts of an estuarine neritid gastropod, Clithon retropictus, and a related marine neritid, Nerita albicilla, were examined under the scanning electron microscope. After ingesting the strain, active responses were seen at the esophagus, stomach and anterior intestine of adult C. retropictus and at the middle and posterior intestines of adult N. albicilla. When the alimentary tracts were isolated from the gastropod and incubated in vitro with strain D3, active response was induced at the most parts of the tract of the adult gastropods and at the stomach and the anterior intestine of juvenile C. retropictus. The responding hemocytes were confirmed to be granulocytes in the semi-thin sections of the tract of adult C. retropictus. The poor hemocyte responses at the middle and posterior intestines of juvenile C. retropictus might support the colonization of the organism there.     

Plasma-dependent chemotactic activity of hemocytes derived from a juvenile estuarine gastropod mollusc, Clithon retropictus, to Vibrio parahaemolyticus and Escherichia coli strains.

Hemocytes of adult Clithon retropictus were attracted chemotactically to live Vibrio parahaemolyticus and Escherichia coli strains. The chemotaxis was stimulated by the plasma of adult C. retropictus. Hemocytes of the juvenile specimen were attracted chemotactically to V. parahaemolyticus and E. coli strains in the presence of the plasma of the juvenile and only to E. coli strain in the absence of the plasma. These evidences suggest that hemocytes of juvenile C. retropictus might be defective to recognize V. parahaemolyticus strains and that the hemocytes would display full activities in the presence of the plasma factor(s).     

Migratory deficiency of Clithon retropictus hemocytes to Vibrio parahaemolyticus and Escherichia coli

Hemocytes of a marine gastropod, Nerita albicilla, but not those of an estuarine gastropod, Clithon retropictus, were observed to migrate to live and heat-killed cells of Vibrio parahaemolyticus and Escherichia coli through Nucleopore membrane in Blind well chamber. The defective migration of C. retropictus hemocytes might reflect, at least in part, the survival of V. parahaemolyticus in the estuarine gastropod.     

Survivals of Vibrio parahaemolyticus and Escherichia coli in a gastropod mollusc, Heminerita japonica.

Vibrio parahaemolyticus strains D-3 and R-13 were found to be cleared within 7 days from a marine neritid gastropod mollusc, Heminerita japonica, maintained in artificial seawater with salinities of 15, 25 and 35 permil (%) at 25 degrees C. Escherichia coli strain YS-2 survived at a level of 10(2) colony forming units per gram in the mollusc maintained in 15% water for up to 14 days and fell to non-detectable level within 7 days in a 35% salinity group. The ability of H. japonica to clear these organisms seems to be less active than that of a marine species. Nerita albicilla, and more active than that of an estuarine species. Clithon retropictus.     

Geographical features of estuaries for neritid gastropods including Clithon retropictus to preserve thermostable direct hemolysin-producing Vibrio parahaemolyticus.

Thermostable direct hemolysin-producing strain of Vibrio parahaemolyticus was not detected from the alimentary tract of 7 neritid gastropods including Clithon retropictus at 9 estuaries of Southwest Islands in Japan in the present study. The strain has been detected from C. retropictus at 2 estuaries facing The Sea of Japan but not at 2 estuaries facing The Seto Inland Sea and The Pacific Ocean in Western Japan in our previous studies. In comparison with geographical features of the estuaries where the strain was detected and not, thick accumulation of muddy sediments at the riverbed and stagnation of brackish water at low tide seem to be essential for the strain to survive in neritid gastropods including C. retropictus.     

Development, characterization, and lethal effect of monoclonal antibodies against hemocytes in an adult female tick, Ornithodoros moubata (Acari: Argasidae)

In the present study, 19 monoclonal antibodies (mAbs) against adult Ornithodoros moubata hemocytes were established, and the reactivity of the hemocytes to these mAbs was examined by an indirect fluorescent antibody test (IFAT), Western blot and immunoprecipitation analyses. It was shown that the reactivities of the hemocytes to the mAbs varied among morphologically similar hemocyte types, and most mAbs produced in the present study showed the multiple band reactivity. However, the presence of shared epitopes among peptide subunits of the same protein or entirely different proteins are not common, so their reactivity could not be explained in detail. These results suggest that there are morphologically similar but functionally differentiated hemocytes. Therefore, in addition to morphological classification, the molecular-based classification of the hemocytes is also required. In order to assess the lethal effect of blood meal containing each mAb, artificial feeding was performed. The OmHC 31 showed the strongest lethal effect on adult female O. moubata. In conclusion, anti-hemocyte mAbs produced in this study are useful not only for the immunological classification of hemocytes but also for the immunological control of the tick.     

Haemophilus parahaemolyticus serotypes. Pathogenicity and cross immunity.

Pigs inoculated intranasally with Haemophilus parahaemolyticus, serotype 2, resisted challenge 3 weeks later with serotypes 2, 4 and 5 without showing clinical symptoms. The pigs were sacrificed 2 days after challenge, and post mortem examination showed a chronic pleuropneumonia from which only serotype 2 was re-isolated. Pigs inoculated intranasally with H. parahaemolyticus, serotype 2, showed no clinical symptoms when challenged 3 weeks later with serotype 1. Post mortem examination revealed a chronic pleuropneumonia with areas of necrosis from which H. parahaemolyticus, serotype 2, was re-isolated, but also small areas of a more acute fibrinous pneumonia from which serotype 1 was re-isolated. The control pig inoculated with only serotype 1 showed a severe acute fibrinous pleuropneumonia. The results indicate that a considerable cross immunity exists between the various serotypes of H. parahaemolyticus.     

Ecological cycle of thermostable direct hemolysin-producing strains of Vibrio parahaemolyticus in a brackish-water area with special reference to molluscs and attached microalgae.

Prevalences of thermostable direct hemolysin (TDH)-producing strains in communities of a gastropod mollusc. Clithon retropictus, and a bivalve mollusc, Corbicula japonica, and levels of the strains in attached microalgae and muddy sediments were investigated at a brackish-water area along Hashizu Creek and Togo Pond in Japan, V. parahaemolyticus was detected from attached microalgae at Hashizu Creek in summer months with the highest level of 1.4 x 10(5) cfu/g. Levels of the organism among 20 animals of C. retropictus and C. japonica at the area varied betwen non-detectable level and 10(3) per mollusc in summer months. TDH was detected from culture supernatants of 11-16% of strains isolated from the algae, sediments and C. japonica and 28% of those isolated from C. retropictus at Hashizu Creek. These evidences suggest that C. retropictus would get TDH-positive strains from the algae.     

Anti-protozoal efficacy of medicinal herb extracts against Toxoplasma gondii and Neospora caninum

The purpose of this study was to determine whether alcohol extracts of herbs (Sophora flavescens Aiton, Sinomenium acutum (Thunb.) Rehder and E.H. Wilson, Pulsatilla koreana (Yabe ex Nakai) Nakai ex T. Mori, Ulmus macrocarpa Hance and Torilis japonica (Houtt.) DC.) from South Korea, possess in vitro anti-protozoal activity against cultures of Toxoplasma gondii and Neospora caninum. These herbs have been used as human anti-parasitics in Asian countries for many years. Alcohol extracts of these herbs were serially diluted to final concentrations ranging from 625 to 19.5 ng/ml in media and added to wells containing either T. gondii or N. caninum tachyzoites in equine dermal (ED) cells. Parasite growth inhibition was measured using 3H-uracil incorporation as compared to untreated controls. T. japonica inhibited T. gondii proliferation by 99.3, 95.5, 73.0 and 54.0% in the range from 156 to 19.5 ng/ml, and S. flavescens inhibited T. gondii proliferation by 98.7, 83.0 and 27.2% in the range from 156 to 39 ng/ml. T. japonica inhibited N. caninum proliferation by 97.8, 97.9, 85.3 and 46.4% in the range from 156 to 19.5 ng/ml. S. flavescens inhibited N. caninum proliferation by 98.6, 97.0, 69.5 and 14.0% in the range from 156 to 19.5 ng/ml. Toxicity to host cells was noted when concentrations of T. japonica and S. flavescens exceeded 625 ng/ml. The herb extracts from S. acutum, Pulsatilla koreana, and U. macrocarpa also showed toxicity at higher levels but did not achieve the same inhibition effects at the lower concentrations against T. gondii and N. caninum as T. japonica and S. flavescens.     

利用不同染料复合染色判定家蚕血细胞的存活状况

家蚕有5种血细胞,可以通过吖啶橙(acridine orange,AO)和碘化丙啶(propidium iodide,PI)染色来区分。溴化噻唑蓝四氮唑[3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide,MTT]是一种广泛应用于细胞增殖和活细胞鉴定的染色剂。通过对家蚕血细胞进行复染,即先用MTT染色,再用AO和PI染色,发现以下2种情况:很多颗粒细胞、浆细胞和所有的原血球细胞不能被MTT染色,但AO染色显示为活的细胞;被MTT染色的细胞表面有紫色纤维状物伸出,除了一部分具有伪足的颗粒细胞外,所有被MTT染色的血细胞也被PI染色,细胞核是红色。因此,利用这3种染料对家蚕血细胞复合染色来判断细胞的存活与死亡,需要仔细分析并分别对待。     

High interferon type I responses in the lung, plasma and spleen during highly pathogenic H5N1 infection of chicken

ABSTRACT: This study shows that high pathogenic H5N1 influenza virus infection of chicken induced high levels of bioactive interferon type I in the lung (4.3 × 105 U/mg tissue), plasma (1.1 × 105 U/mL), and spleen (9.1 × 105 U/mg tissue). In contrast, a low pathogenic attenuated H5N1 vaccine strain only induced approximately 24 times less IFN in the lung, 441 times less in the spleen and 649 less in the plasma. This was in the same range as a reassortant carrying the HA from the vaccine strain and the remaining genes from the high pathogenic virus. On the other hand, a reassortant virus with the HA from the high pathogenic H5N1 with the remaining genes from the vaccine strain had intermediate levels of IFN. The level of interferon responses related to the viral load, and those in the spleen and blood to the spread of virus to lymphoid tissue, as well as disease severity. In vitro, the viruses did not induce interferon in chicken embryonic fibroblasts, but high levels in splenocytes, with not clear relationship to pathogenicity and virulence. This, and the responses also with inactivated viruses imply the presence of plasmacytoid dendritic cell-like leukocytes within the chicken immune system, possibly responsible for the high interferon responses during H5N1 infection. Our data also indicate that the viral load as well as the cleavability of the HA enabling systemic spread of the virus are two major factors controlling systemic IFN responses in chicken.     

Virus characterization, clinical presentation, and pathology associated with H7N3 avian influenza in British Columbia broiler breeder chickens in 2004

Low-pathogenicity avian influenza (LPAI) subtype H7N3 was diagnosed on a two-age broiler breeder farm in Abbotsford, British Columbia (BC), in early February 2004. The presenting complaint in the older index flock was feed refusal, with 0.5% mortality over 72 hr that resolved over the following week Ten days after the initial complaint in the index flock, a younger flock in an adjacent barn experienced an abrupt spike in mortality (25% in 48 hr). The gross lesions of tracheal hyperemia and hilar pulmonary consolidation were subtle and nonspecific, and the diagnosis of avian influenza required laboratory confirmation. Two different viruses were isolated from the index farm: a LPAI (H7N3) was isolated from the older flock and a high-pathogenicity avian influenza (HPAI) (H7N3), which had an additional 21 base insertion at the hemagglutinin-cleavage site, was isolated from the younger flock. The presence of this insertion sequence and the similarity of adjacent sequences indicate that the LPAI had mutated into HPAI at some point between the first and second barn. Despite enhanced on-farm biosecurity measures, the virus was not contained on the index farm and eventually spread to over 40 commercial poultry facilities before massive depopulation efforts enabled its eradication.     

金色葡萄球菌、伤寒沙门氏菌和副溶血弧菌单管多重PCR检测方法的建立及初步应用

本研究根据金黄色葡萄球菌（Staphylococcus auretls，SA）耐热核酸酶（nut）基因、伤寒沙门氏菌（Salmonella typhi，ST）鞭毛抗原（H1-d）基因和副溶血性弧菌（Vibrio parahaemolyticus，VP）热稳定直接溶血素（tdh）基因，分别设计了三对引物Nuc-F／Nu-R、H1-d—F／H1-d—R和Tdh-F／Tdh-R，预计PCR扩增的DNA片断分别为279bp、202bp和458bp。通过对单个基因PCR和单管多重PCR扩增的特异性、敏感性分析以及对单管多重PCR扩增条件如引物浓度、退火温度和dNTP浓度等的优化，建立了快速检测金黄色葡萄球菌、伤寒沙门氏菌和副溶血弧菌的单管多重PCR方法，该方法检测的敏感性分别为：47．8PgST的基因组DNA．22．7PgSA的基因组DNA和0．3745PgVP的基因组DNA。模拟试验显示最低检测限度为分别为：SA150CFU／反应体系，ST98CFU／反应体系，VP53CFU／反应体系。表明该方法具有很好的应用价值和开发前景。     

结缕草根际联合固氮菌的分离及初步鉴定

联合固氮菌在植物根际土壤生长或定植于植物根表细胞,这类细菌可将空气或土壤中的氮素转化为氨态氮供植物直接吸收利用.结合气相色谱仪,利用乙炔还原等方法对结缕草根际联合固氮菌进行了分离和鉴定.结果表明:从结缕草根际土壤中分离获得9株联合固氮菌株,菌株的固氮酶活性相差较大,为67.5～343.7 C2H4nmol/(ml·h)...     

外源甜菜碱对低温胁迫下结缕草生理特性的影响

甜菜碱作为一种重要的渗透调节物质,与低温胁迫下植物的抗逆性有着密切的联系。为了探讨不同浓度外源甜菜碱对低温胁迫下结缕草相关生理指标的影响,本实验以结缕草‘青岛’品种为实验材料,分别采用0,50,100和150 mmol/L 的甜菜碱进行叶面喷施,在8/2℃(白天/夜间)的培养箱中连续处理28 d,以正常管理(28/24℃)作为对照。结果表明:外源甜菜碱能够有效缓解低温胁迫下结缕草坪观质量和叶绿素含量的下降,减少电解质渗透率和丙二醛含量的升高,显著提高SOD、POD、CAT和APX的活性,从而降低H₂O₂和O2·-的累积,同时,外源甜菜碱还可以提高低温胁迫下结缕草可溶性蛋白和脯氨酸的含量,增强渗透调节能力,进而增强结缕草对低温胁迫的抵抗性。其中,外施100 mmol/L甜菜碱对提高结缕草的耐低温能力的效果最为显著。     

Serological and immunological studies of pleuropneumonia of swine caused by Haemophilus parahaemolyticus

The development of circulating antibodies for H. parahaemolyticus was studied in experimentally infected SPF pigs and in-contact SPF pigs. Blood serum titers were determined by a modified complement fixation test with normal SPF swine serum as a source of supplementary complement factor, and by an indirect haemagglutination test.CF and IHA titers became positive within the first 2 weeks following exposure to H. parahaemolyticus, and reached peak values after 2 to 7 weeks (Figs. 1 to 3). The exposed pigs proved immune, in that they showed no clinical symptoms on challenge after resp. 6, 9 and 11 weeks.While distinct titers were thus obtained with both tests in SPF swine experimentally exposed to H. parahaemolyticus, the CF test proved more specific than the IHA test when the 2 tests were compared in a field outbreak of polyserositis (Glässers disease) caused by H. parasuis. The CF test would therefore seem to be preferable to the IHA test in field diagnostic work (Table 1).A noticeable finding was that challenge did not elicit an anamnestic antibody response in any of the immune pigs (Figs. 1 to 3). This fact together with negative bacteriological findings in the animals in question would seem to suggest that the challenge dose was unable to establish a permanent infection in the respiratory tract of the immune pigs.     

Genetic-diet interactions in the Hyperkalemic Periodic Paralysis syndrome in Quarter Horses fed varying amounts of potassium: I. Potassium and sodium balance, packed cell volume and plasma potassium and sodium concentrations

Six broodmares that were genetically tested to be heterozygous (H/N) and six broodmares that were tested to be homozygous negative (N/N) for Equine Hyperkalemic Periodic Paralysis (HYPP), that were descendents of the same stallion, were used in a replicated 3×3 Latin square experiment to determine the genetic-diet relationships in the HYPP syndrome.¹ The mares were fed rations consisting of 65% pelleted concentrate and 35% Coastal Bermuda grass hay that provided 1.1 (diet A), 1.9 (diet B) and 2.9% (diet C) potassium. The experimental periods were 14 days long, resulting in 28 meals per period. At meals 1 and 27, blood samples were taken every 30 minutes for 12 hours and every 10 minutes from 2 to 5 hours post-feeding. Total urine and fecal collections were made on the last 4 days of each period.Water, feed and fecal samples were analyzed for sodium and potassium content. Blood was analyzed for packed cell volume (PCV) and plasma potassium (K⁺) and sodium (Na⁺) concentrations.Apparent absorption of potassium was 99.8% across all diets, and potassium was excreted principally in the urine. More potassium was retained when the horses were fed diet B than diets A or C. Apparent absorption of sodium was 99.6% with no difference by diet. Neither potassium nor sodium balances were affected by HYPP status.There was no post-prandial pattern of plasma K⁺ concentration seen when the horses were fed the low potassium diet. When fed the higher potassium diets, they had greater plasma K⁺ values and exhibited a post-prandial peak at 2 to 5 hours after feeding. This peak occurred in all of the horses, meaning that plasma K⁺ cannot be used as a diagnostic aid for the presence of the HYPP mutation. By meal 27, some adaptation had occurred resulting in lower plasma K⁺ values when the horses were fed the higher potassium diets. The horses had increased plasma K⁺ concentrations throughout the post-prandial period while fed diet B compared to diet A, agreeing with the positive potassium balance seen in this experiment. The HYPP H/N horses had lower plasma Na⁺ concentrations in the early post-prandial hours and continuously greater PCV values than the HYPP N/N horses.     

Effect of Haemonchus contortus infection on the clearance of antipyrine, sulfobromophthalein, chloramphenicol, and sulfathiazole in lambs

A study was made to determine the effect of Haemonchus contortus parasitic infection in lambs on the clearance of several IV administered drugs. Clearance of sulfobromophthalein or sulfathiazole from the plasma of lambs was unaffected by infection with H contortus. Clearance of antipyrine was enhanced by the infection, and thiabendazole treatment did not alter this effect. Clearance of chloramphenicol (CAP), administered as the succinate ester (CAPS), was not changed by the infection, but it was increased after treatment with thiabendazole. Changes in the mean body residence time and initial plasma concentration of CAPS and CAP after treatment with thiabendazole indicate that hydrolysis of CAPS to CAP was reduced. High concentrations of CAPS apparently enhanced its own elimination directly rather than via the expected sequence involving hydrolysis, glucuronidation, and excretion of CAP-glucuronide. Enhanced clearance of antipyrine following infection of lambs with H contortus can be explained in at least 2 ways. First, it is possible that the lambs did not have mature amounts of hepatic drug metabolizing enzyme activity as reported by other investigators, which may be explained by breed differences or animal husbandry practices. Second, infection of lambs by H contortus may have triggered an inductive response in hepatic cytochrome P-450-mediated activities, which might result via a generalized enhancement in hepatic protein synthesis associated with the physiologic response to replace plasma proteins and other blood components lost through gastrointestinal hemorrhage caused by the active feeding of adult worms. Other phase-II reactions such as acetylation, glucuronidation, and glutathione-S-transferase apparently were not affected.