Immunoenzyme method for detecting microbial producers of palytoxin

A competitive ELISA using the intact toxin as a coating antigen for detecting palytoxin was developed. This immunoassay allows palytoxin (PTX) to be determined in the range of 6-250 ng/ml. In sensitivity, this determination is comparable with RIA but is three times inferior to ELISA using monoclonal antibodies. Inhibition experiments using some toxins of marine invertebrates proved the serological specificity of the palytoxin binding to antibodies. Both the indirect and competitive ELISA were used to find PTX-producing bacteria among 420 isolates of sea bacteria. It was found that gram-negative bacteria Aeromonas sp. and Vibrio sp. associated with toxic samples of the soft coral Palythoa sp. produced compounds antigenically related to PTX.     

Development and Comparative Evaluation of a Competitive ELISA with Rose Bengal Test and a Commercial Indirect ELISA for Serological Diagnosis of Brucellosis

The development of a competitive ELISA for the detection of brucella-specific antibodies in bovines is described. Anti-brucella guinea pig serum was used as a source of competing antibodies. Lipo-polysaccharide purified from inactivated B. abortus S19 culture was used as antigen for the development of the assay. Sera from cattle were used in the competitive ELISA, rose bengal test and a commercial indirect ELISA. The following cattle sera were tested: (i) known positive sera (n = 80) (ii) known negative sera (n = 100) and (iii) field sera (n = 1184). Based on the receiver operating characteristics curve analysis and frequency distribution of the percentage of inhibition, 30% inhibition was considered the cut-off for positive and negative results. The sensitivity and specificity estimate on comparison with the commercial indirect ELISA was 94.87 and 92.12% respectively. The competitive ELISA described is a simple method for the routine screening of animal sera for detecting Brucella-specific antibodies.     

Studies on Endophytic Colonization Ability of Two Upland Rice Endophytes, <Emphasis Type="Italic">Rhizobium</Emphasis> sp. and <Emphasis Type="Italic">Burkholderia</Emphasis> sp., Using Green Fluorescent Protein Reporter

Colonization ability of the two endophytic bacteria, isolated from surface sterilized roots of upland cultivated rice viz., Rhizobium sp. and Burkholderia sp., was compared after genetically tagging them with a constitutively expressing green fluorescent protein gene (gfp/gusA). Confocal laser scanning microscopy (CLSM) of gnotobiotically grown seedlings of Narendradhan 97, inoculated with gfp/gusA-tagged endophytes, revealed that both Rhizobium sp. and Burkholderia sp. colonized the intercellular spaces in the root cortex when inoculated separately. Colonization by gfp/gusA-tagged Rhizobium sp. was severely inhibited when co-inoculated with an equal number (10⁶ cfu ml⁻¹) of wild type Burkholderia sp. Burkholderia sp. was a more aggressive endophytic colonizer of rice than Rhizobium sp. The potential of using gfp/gusA reporter and CLSM as tools in evaluating competitive ability of colonization among endophytes is demonstrated in this study.     

FIRST EVIDENCE OF PALYTOXIN ANALOGUES FROM AN OSTREOPSIS MASCARENENSIS (DINOPHYCEAE) BENTHIC BLOOM IN SOUTHWESTERN INDIAN OCEAN1

Benthic dinoflagellates of the genus Ostreopsis Schmidt are common in tropical and subtropical water, and some species produce toxins potentially involved in human intoxication events. A benthic bloom of Ostreopsis mascarenensis Quod was observed near Rodrigues Island during a survey of benthic dinoflagellates in the southwestern Indian Ocean. The morphology of O. mascarenensis was studied by LM and SEM. Preliminary screening of a crude extract of an O. mascarenensis bloom revealed neurotoxicity in mice similar to that induced by palytoxin. After partition of the crude extract, the highest toxicity was retained in the butanol‐soluble fraction, which retained hemolytic activity suggestive of palytoxin analogues. Two new toxins, mascarenotoxin‐A and ‐B, were resolved from this fraction by HPLC coupled to a diode array detector. The closed mass spectrum profile and fragmentation pattern obtained by advanced nano–electrospray ionization quadrupole time‐of‐flight mass spectrometry between purified toxins and a reference palytoxin confirmed the mascarenotoxins as palytoxin analogues. These results were confirmed by tandem mass spectrometry with the identification of specific fragment ion m/z 327. An on‐line liquid chromatography protocol coupled to tandem mass spectrometry was developed for detection of these palytoxin analogues. The present study describes the first purification, chemical, and toxicological characterization of new palytoxin analogues isolated from a benthic bloom of O. mascarenensis. These results suggest that O. mascarenensis, which is largely distributed in the southwestern Indian Ocean, could be a source of palytoxin poisoning in this tropical area.     

Serological Studies of an Acid-Labile O-Polysaccharide of Proteus vulgaris OX19 Lipopolysaccharide Using Human and Rabbit Antibodies

In a Weil-Felix test, sera from patients infected with Rickettsia sp. agglutinate Proteus OX types of bacteria and Proteus lipopolysaccharide (LPS) are responsible for the cross-reaction. Data on the character of LPS of one of the OX group strains, Proteus vulgaris OX19, are contradictory, and it remained unclear whether it has an O-polysaccharide (OPS) and is thus LPS of the smooth type (S) or not (rough-type LPS). Our studies showed that P. vulgaris OX19 (strain PZH-24) produces a smooth-type LPS that contains a long-chain OPS, but it undergoes depolymerization during mild acid hydrolysis conventionally used for LPS delipidation and loses the serological activity. An elucidation of the complete structure of OPS demonstrated the presence of a glycosyl phosphate linkage responsible for the acid-lability of the polysaccharide chain. In ELISA, both IgM type antibodies in a Weil-Felix test with human anti-Rickettsia typhi sera and rabbit anti-P. vulgaris OX19 antibodies reacted with OPS. Rabbit antibodies did not inhibit the cross-reaction with human antibodies and thus bind to different epitopes.     

A Specific Method for the Detection of Hiochi Bacteria Using an Enzyme-linked Immunosorbent Assay System with Anti-hiochi Bacteria Monoclonal Antibodies

Ten standard strains of hiochi bacteria were selected based on the SDS-PAGE patterns of their cellular proteins. We then obtained ten hybridoma systems that secreted highly reactive monoclonal antibodies (MAbs) to the whole cells of each strain. It was apparent that these MAbs were highly reactive and specific for hiochi bacterial whole cells when using an enzyme-linked immunosorbent assay (ELISA). Three MAbs (two against the homo-fermentative hiochi lactobacilli and an MAb against the hetero-fermentative true hiochi bacilli) showed cross-reactivity to some of the other strains of lactobacilli tested. However, the other MAbs did not react with strains other than the immunogen. A sensitive ELISA method for the detection of hiochi bacteria was examined. It was possible to detect the order of 10³ cells of the ten standard strains. Using this procedure and a mixture of the ten MAbs, a detection limit of 10⁴ cells or less could be obtained for 98.2% of the hiochi bacteria isolated from sake brewing factories. Thus, this immunological technique using MAbs specific for hiochi bacteria is a sensitive and rapid detection method for hiochi bacteria, which can be used in the quality control of sake.     

Pathogenic bacteria associated with lesions and thallus bleaching symptoms in the Japanese kelp Laminaria religiosa Miyabe (Laminariales,Phaeophyceae)

During early Spring (April–May) when the seawater salinity drops suddenly and the seawater temperature increases drastically, severe lesions and thallus bleaching were observed in the Laminaria religiosa population at Oshoro Bay, Otaru, Hokkaido, Japan. The healthy and diseased kelp blades were collected and subjected to enumeration of total number of culturable bacteria and bacterial species. Bacterial enumerations were done using 3 different media formulations; high-nutrient media (Media 1), low-nutrient media (Media 2) and modified low-nutrient media with 5% kelp extract (Media 3). Seven bacterial species were isolated from the healthy kelp. These were Alcaligenes aquamarinas, Alteromonas sp., Azomonas agilis, Azotobacter beijerinckii, Escherichia coli, Halobacterium sp. and Halococcus sp. All 7 bacterial species were isolated on Media 2 and Media 3, but only 5 species were isolated using Media 1 with the absence of Halobacterium sp. and Halococcus sp. Highest total number of culturable bacteria was 2050 CFU/cm² on Media 3. Eight species of bacteria were isolated from the diseased kelp thallus with the addition of Erwinia amylovora. All 8 bacteria grew on Media 2 and Media 3, but only 6 species were isolated using Media 1 with the absence of Halobacterium sp. and Halococcus sp. Highest total number of culturable bacteria was 5830 CFU/cm² on Media 3. However, only 3 species were isolated from the lesioned area. The most abundant species was Alteromonas sp. followed by Halococcus sp. and Alcaligenes aquamarinas. The surface bacteria showed best growth on Media 3. Scanning Electron Microscopic images of the healthy and diseased thallus gave distinctive evidence of the severity of the lesions as well as the relative abundance in the bacterial population. In an effort to identify the symptoms causative organism, the isolated bacterial species were cultured and used to test Koch's postulates. Out of the 8 species, only Alteromonas sp. induced lesions on the axenic kelp blades. The inoculated bacteria were also re-isolated without any significant contamination. Hence, Alteromonas sp. is suggested as the possible disease causing organism.     

Investigation of microcystin removal from eutrophic surface water by aquatic vegetable bed

In southern China, many freshwater ecosystems, including lakes, rivers and reservoirs, are eutrophic. The nutrient loading coupled with year-round warm weather favors the growth of cyanobacteria, several of which can produce cyanotoxins, especially the potent liver toxins called microcystins, which are often detected in eutrophic drinking water sources. For purifying raw water used as source of drinking water treatment plants, an aquatic vegetable bed (AVB) experiment had been carried out in a hypertrophic waterfront of Lake Taihu, China, since October 2002. AVB was a simplification of the nutrient film technique (NFT) used to produce vegetables, which requires large quantities of water and nutrients. The average removal rates of total microcystin-RR and microcystin-LR were 63.0% and 66.7%, respectively. This study indicated that Ipomoea aquatica was able to absorb microcystins by using enzyme-linked immunosorbent assay (ELISA), and that the roots absorbed more toxins than leaves and stems. We used fluorescence in situ hybridization (FISH) to analyze the density of microcystin-degrading bacteria in AVB sediment. Two species of microcystin-degrading bacteria were detected, which indicated that microcystin bio-degradation processes did occur in AVB. Protozoa and metazoa were abundant in the rhizosphere. Aspidisca sp., Vorticella sp., Philodina sp., and Lecane sp. were the dominant species. The predation function of protozoa and metazoa had a positive effect on removal of cyanobacteria and microcystins.     

Isolation and characterization of heavy metals resistant bacteria from Lagos Lagoon

A total of 228 bacteria with an ability to resist toxic heavy metals were isolated from 8 selected sites of the Lagos Lagoon. The bacteria isolated wereStaphylocaccus sp.,Bacillus sp.,Pseudomonas sp.,Streptococcus sp.,Moraxella sp.,Escherichia coli, Proteus sp.,Klebsiella sp. andSalmonella sp. The heavy metals to which resistance was recorded were mercury, lead, zinc, cobalt, copper and chromium. The lagoon sites from which the highest number of resistant bacteria were isolated were Marina and Ebute-Ero. The heavy metal to which most bacteria were resistant was cobalt, while the least was chromium. The significance of the result is discussed in relation to the Nigerian environment and human health.     

Preparation of fluorescent labeled nodule bacteria strains of wild legumes for their detection in vivo and in vitro

A series of expression vectors containing TurboGFP and TurboRFP genes of fluorescent proteins under the control of the T5 phage constitutive promoter was created for a vital staining of nodule bacteria. These vectors were either obtained using the broad host range pBBRI replicon for labeling of strains, where a marker gene was expressed from a transformed plasmid, or they were prepared using the pRL765 gfp plasmid for labeling of strains via the introduction of genes of fluorescent proteins into the bacterial chromosome. Transformation was shown to be the most convenient method of transfer of constructions into cells of nodule bacteria, as there exists the possibility of spontaneous plasmid mobilization and, consequently, its transition from cells of labeled strains into other soil bacteria if the mob locus is present in vectors needed for conjugation. Fluorescent labeled strains of Rhizobium sp., Mesorhizobium sp., Ensifer (Sinorhizobium) sp., Bradyrhizobium sp., Phyllobacterium sp., and Agrobacterium sp. were prepared using the obtained vector constructions. The suitability of the obtained strains for both in vivo and in vitro experiments was demonstrated.     

Use of cluster analysis with monoclonal antibodies for taxonomic differentiation of phytopathogenic fungi and for screening and clustering antibodies

Cluster analysis of ELISA (A405) values resulting from tests using all possible combinations of 43 monoclonal antibodies (raised againstPhytophthora megasperma f.sp.glycinea) and 14 phytopathogenic fungi separated these fungi into taxonomically appropriate clusters. Two distance measurements (Pearson's correlation coefficient and normalized Euclidean distance) and five linkage methods (single, complete, median, centroid, and average) were evaluated. The combination of Pearson's correlation coefficient and complete linkage produced the most natural clusters. A modification of the data matrix allowed clustering of the monoclonal antibodies by specificity. Numerically coded ELISA values clustered identically with the orginal data. The taxonomic and evolutionary implications of using monoclonal antibodies specific to carbohydrates are discussed.     

Evaluation of an enzyme-linked immunosorbent assay (ELISA) for the detection ofCampylobacter jejuni antibodies,and comparison with a complement fixation test (CFT)

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of total anti-Campylobacter immunoglobulins in human sera. In this assay disintegratedCampylobacter bacteria were used as the antigen. Absorption tests including other possibly enteropathogenic bacterial species showed that the ELISA system displayed a high immunological specificity forCampylobacter. Using this ELISA it was found that in about 80% ofCampylobacter patients theseCampylobacter antibodies are produced to almost maximal levels within 8 days after onset of disease, and that they may persist for at least 4 months. Indeed,Campylobacter antibodies were demonstrated at low levels in a large number of control sera. However, accepting an antibody titre of 1: 640 as indicative ofCampylobacter infection, the statistical sensitivity of the ELISA system was 77% and the specificity 95%. In an epidemiological survey a high association was demonstrated between the severity ofCampylobacter-related symptoms and antibody titre values. Assessment ofCampylobacter antibody titres by means of this ELISA and by a complement fixation test in 92 sera from index patients and contacts with and without symptoms showed a high association of results.     

Interspecific competitive ability of homokaryotic and heterokaryotic wood decay basidiomycetes

The role of the homokaryotic life stage in the dynamics of fungal communities is relatively unknown. However, homokaryons are thought to be only a temporary stage and are therefore not generally used in ecological experiments with fungi. In this study, the relative competitive ability and growth rates of homokaryons and heterokaryons of wood decay fungi were tested to assess the potential role of homokaryons in community dynamics. A homokaryon and a heterokaryon of each of four species (Aleurodiscus lividocoeruleus, Peniophora sp. 1, Peniophora sp. 2 and Pereniporia medulla‐panis) were assessed for their competitive abilities on an agar medium. The relationship between nuclear status and competitive ability varied between species. The homokaryon of Peniophora sp. 2 was competitively superior to its heterokaryon, whereas the homokaryon of Peniophora sp. 1 was inferior to its heterokaryon. A hierarchy of competitive abilities of each isolate revealed that Pereniporia medulla‐panis homokaryon = P. medulla‐panis heterokaryon > Peniophora sp. 1 heterokaryon > Peniophora sp. 2 homokaryon > Peniophora sp. 2 heterokaryon > A. lividocoeruleus heterokaryon = A. lividocoeruleus homokaryon. This experiment indicates that homokaryons as well as heterokaryons have the potential to influence community structure through competitive effects.     

乳房链球菌GapC蛋白的表达及其B细胞抗原表位的预测与鉴定

乳房链球菌Streptococcus uberis的GapC蛋白是一种位于该菌表面的具有甘油醛-3-磷酸脱氢酶活性的蛋白,其参与细胞活动,表现出多种生物学活性,此外还具有良好的抗原性。文中旨在对乳房链球菌GapC蛋白可能的B细胞抗原表位进行预测,分析和验证候选表位肽的免疫原性。利用S. uberis分离株RF5-1克隆gapC基因,构建重组表达质粒pET-28a-GapC,诱导表达GapC重组蛋白,并以纯化蛋白免疫家兔,获得抗GapC多抗。利用生物信息学软件预测并分析GapCB细胞抗原表位的三维结构和空间位置及对GapC蛋白及表位的同源性比较。结果表明,表达纯化了44kDa的GapC蛋白具有良好的反应性。利用表位预测软件筛选并合成针对S.uberisGapC蛋白的6个线性和3个构象优势B细胞表位多肽,三维结构的分析显示,筛选的多肽具有良好的抗原表位形成条件。以纯化的S.uberis GapC蛋白免疫家兔制备多抗,通过间接ELISA对抗原表位进行鉴定。ELISA检测结果显示,9条抗原表位肽均可不同程度地与抗GapC多抗反应,其中表位266AANDSYGYTEDPIVSSD282与多抗反应...     

Immunological quantification of recA protein in cell extracts ofE. coli after exposure to chemical mutagens or UV radiation

Increased synthesis of RecA protein is induced inE. coli cells after their damage, the rate of synthesis being dependent on the extent of DNA alterations. The level of the RecA protein was determined inE. coli cell extracts after damage induced by NQO, MNNG, MMC, NAL or UV radiation, using competitive enzyme-linked immunosorbent assay (ELISA). PurifiedE. coli RecA protein and rabbit monospecific polyclonal antibodies against it were prepared for the quantitative assay. The level of theRecA protein was increased after treatment with all mutagens. Contrary to other induced proteins, the synthesis of the RecA protein increased within 30 min after damage with UV radiation at a relatively slow rate. The ELISA method made it possible to determine 0.5–50 ng of the RecA protein in bacterial extracts. The method can be employed as an auxiliary test for DNA damage determination and also in studied concerning the role of the RecA protein in repair processes. Translated by I. Miler     

The production and utilisation of monoclonal antibodies for identification of a Frankia strain utilised as inoculum for Casuarina equisetifolia

Monoclonal antibodies were raised against Frankia 0RS020607, a strain isolated originally by H.G. Diem from nodules of Casuarina equisetifolia from Senegal. One of these antibodies, mAb8C5, was shown by ELISA to have high, but not absolute specificity for 0RS020607. This antibody was employed to investigate the mobility and persistence of 0RS020607 in plantations of C. equisetifolia. Seedlings were inoculated in pots of sand in a forest nursery with 0RS020607, with local crushed nodule suspensions or were left uninoculated. They were planted out after 5 months in experimental plots on a moderately fertile black soil site and on a low organic, oxidised red soil site. Compared with crushed nodule inoculated seedlings or uninoculated controls, growth of seedlings at transplant was improved by inoculation with Frankia 0RS020607. However, 4 years after transplant to experimental plots, the growth of trees receiving different treatments was similar. The possibility that movement of ORS 020607 between treatment plots contributed to new nodulation and enhanced growth of uninoculated trees was tested using mAb8C5 in ELISA of Frankia mycelium, extracted from the nodules of trees of the three treatments. No significant differences in reactivity were detected between nodules from uninoculated and 0RS020607 inoculated trees at either the black or the red soil sites, showing that 0RS020607 moved between treatment plots at both sites. However, at both sites, nodules from plots of trees that were inoculated originally with local crushed nodules gave reactions in ELISA that were significantly lower than values for 0RS020607 inoculated trees, possibly due to the competitive effects for new nodulation of enhancement of the indigenous population of Frankia. Serological techniques using antibodies of high specificity against Frankia strains have value for rapid screening of field samples as a preliminary for further analysis by more discriminatory techniques based on assays of genetic polymorphisms.     

Detection of hemolytic bacteria from <Emphasis Type="Italic">Palythoa caribaeorum</Emphasis> (Cnidaria,Zoantharia) using a novel palytoxin-screening assay

Palytoxin (PTX), one of the most potent and chemically complex marine toxins, is predominantly found in zoanthid corals and sporadically in dinoflagellates. Its biosynthesis and metabolic pathways are largely unknown. However, the widespread occurrence of the toxin in phylogenetically distinct marine organisms is consistent with its production by microorganisms and subsequent accumulation in the food chain. To investigate a possible microbial origin, bacteria from two zoanthid corals (Palythoa caribaeorum, Zoanthus pulchellus) and one sponge (Neofibularia nolitangere) were isolated. More than 250 bacteria were screened for hemolysis using a newly developed PTX-screening assay of which 7% showed PTX-like hemolytic activity. 16S rRNA gene sequencing revealed that these bacterial isolates belonged to strains of Bacillus cereus group (n = 11) as well as the genera Brevibacterium (n = 4) and Acinetobacter (n = 2). The results indicate the presence of Na⁺/K⁺-ATPase toxins and possibly PTX in hemolytic bacteria from P. caribaeorum.     

Neutralization of Chlamydia psittaci with Monoclonal Antibodies

Neutralization of Chlamydia (C.) psittaci avian strain P-1041 was examined in vitro using monoclonal antibodies (MAbs). Of the 10 MAbs used, 6 were found to exhibit neutralizing capability. These include 3 against major outer membrane protein (MOMP), 1 against lipopolysaccharide (LPS) and 2 against other protein molecules [90 kilodalton (kDa) and 90/50 kDa]. Most neutralizing MAbs were dependent on complement for efficient neutralization, while a strain-specific MAb (2B5) against the 90 kDa protein displayed a different requirement for complement and neutralized the infectivity of the P-1041 at high concentrations without complement. By competitive inhibition enzyme-linked immunosorbent assay (competitive inhibition ELISA), all 3 neutralizing anti-MOMP MAbs were demonstrated to recognize different epitopes found in very close proximity to each other on the outer membrane.     

Changes in the Antigenic Properties of Azospirillum brasilense Lipopolysaccharide,Induced by Addition of Tris-(hydroxymethyl)-aminomethane into the Culture Medium

Addition of tris-(hydroxymethyl)-aminomethane (Tris) into the culture medium of Azospirillum brasilense sp245 changes the antigenic properties of the lipopolysaccharide (LPS) isolated from the external membrane of the bacteria. LPS preparations from the bacteria grown in the presence of Tris have been analyzed by immunodiffusion, using monospecific antibodies. The disappearance of the precipitation band corresponding to one of the two O-specific polysaccharides of the LPS (O-PS) and changes in the electrophoretic profile have been revealed. However, only minor differences in absorption spectra of products of O-PS1 reaction with phenol/sulfuric acid have been demonstrated between the bacteria grown under standard conditions and in the presence of Tris.     

Alkalobacter lublini gen. nov., sp. nov.

On the basis of phenotypic properties and G+C content of DNA, as well as competitive DNA-DNA hybridization and extracellular polymeric substance analysis it was shown that this strain was completely different from all other alkaliphilic bacteria. We hereby propose that this strain be designatedAlkalobacter lublini gen. nov., sp. nov.