Gel Double Diffusion Research Articles

Comparisons of some properties of two laboratory variants of Raspberry bushy dwarf virus (RBDV) with those of three previously characterised RBDV isolates

The properties of two laboratory variants of Raspberry bushy dwarf virus (RBDV), genus Idaeovirus, were compared with those of their parental sources and with two naturally occurring variants. Isolate RB is a natural variant able to overcome the resistance to RBDV present in some red raspberry cultivars. Isolate M is a serological variant from black raspberry. Laboratory variant D1, was derived from the Scottish type isolate (D200) by continuous sub-culture in Chenopodium quinoa. Laboratory variant Can-S was derived from an isolate infecting Canby red raspberry in Canada (Can) after passage through Nicotiana benthamiana. All isolates reacted with a polyclonal antiserum to isolate D200 in agarose gel double-diffusion tests but, whereas isolates D200, RB, Can and Can-S were serologically indistinguishable, the precipitin lines formed by these isolates each spurred over those formed by isolates D1 and M. All six isolates reacted strongly with the polyclonal antiserum in double antibody sandwich and plate-trapped antigen (PTA) forms of ELISA and in Western blotting (WB) and when each of four monoclonal antibodies (Mabs) to an unnamed red raspberry isolate from Canada was used to detect antigen trapped by the polyclonal antiserum. However, the virus isolates differed in their reactions to these four Mabs in PTA-ELISA and in WB. Isolates RB, Can and Can-S behaved similarly in these tests as did isolates D200 and D1, but isolate M was distinct. In herbaceous test plants, variants D1 and Can-S were readily distinguished from their parental sources and from the other two isolates by producing either no symptoms (D1) or very severe symptoms (Can-S) in hosts. Unlike all other isolates studied world-wide, Can-S failed to infect C. quinoa systemically but induced severe necrotic local lesions in this and other hosts. Reverse transcription-polymerase chain reaction was used to amplify the gene encoding the coat protein (CP) in RNA-2, and a region of the gene encoding the polymerase in RNA-1. The nucleotide sequences of the CP genes of the six isolates were > 96% identical but isolate Can-S was the most distinctive. However, the similarity between Can-S and its parent isolate (Can) was no greater than the similarity between Can-S and the other isolates, suggesting that Can-S may not have arisen as the result of a mutation from isolate Can. Sequence comparisons of parts of the polymerase gene of isolates R15, D1, D200 and Can-S showed that they were 95–98% identical.

Serological and biochemical distinguishing of Pseudomonas syringae pathovars on peas

Polyclonal antisera to detect and determine two related pathovars, Pseudomonas syringae pv. pisi and Pseudomonas syringae pv. syringae, were prepared Untreated bacterial cells, or fixed by formaldehyde or glutaraldehyde were used as antigens. After cross-absorption with heterologous antigens, antisera revealed a high level of specificity in slide agglutination, Ouchterlony gel double-diffusion and in DAS-ELISA and PTA-ELISA. Each of 14 P s. pisi prepared polyclonal antisera could detect and deter­ mine all strains of P s. pisi, regardless of race. PTA-ELISA was the most appropriate serological test to distinguish Pseudomonas syringae pathovars on peas. Most of the P s. pisi strains from foreign collections were in serological tests confirmed asP. s. pisi, while most of the Czech strains suspected as P s. pisi were determined as P s. syringae strains. The principal biochemical reaction, i.e., use of DL-homoserine as a carbon source to grow P s. pisi but not P s. syringae , was proved not to be sufficiently reliable to distinguish both pathovars.

Characterization of Shigella dysenteriae type 1 capsular polysaccharide by immunochemical methods.

We have isolated the capsular polysaccharide from the strain of Shigella dysenteriae type 1 8337. The product was purified by ultracentrifugation, treated with enzymes (proteinase K, DNA-RNAase) and analyzed by immunochemical methods. Polyclonal antibodies were obtained from rabbits immunized by whole cell antigens prepared from Shigella by ultrasonic treatment and by purified capsular polysaccharide. Crossed immunoelectrophoresis, PAGE and Western blot analysis showed that this product containing mainly the polysaccharide component also contained glycoprotein and lipopolysaccharide. Double diffusion in agarose gel confirmed that the capsular preparation contained at least three antigens reacting with rabbit polyclonal antiserum.

Prevalencia serológica y evaluación de los factores de riesgos de leucosis bovina enzoótica en la Sabana de Bogotá y los Valles de Ubaté y de Chiquinquirá, Colombia

The authors present the results of a study to determine the serological prevalence of the enzootic bovine leukosis virus among dairy cows in the Sabana de Bogotá region and the Ubaté and Chiquinquirá Valleys, the principal dairying areas of Colombia. Samples were taken from 919 animals selected at random from 420 farms in 51 municipalities, in accordance with a statistical sampling procedure developed previously, based on the cattle census maintained by the Animal Health and Disease Control Office of the Instituto Colombiano Agropecuario, and the recommendations for prevalence studies of the Pan American Health Organization. The double gel diffusion technique with gp-51 antigen was used. Serological prevalence averaged 45.28% (ranging from 42.07% to 48.49%) with a confidence level of 95%. In addition, a survey was conducted to determine how much those in charge of herds knew about the disease, and to establish the incidence of certain risk factors possibly associated with distribution of the causal agent. Data obtained from 232 completed questionnaires showed that only 54.6% of farms received professional assistance. Of these, 6.6% received constant assistance, 4.4% received visits every fortnight, 51.8% received monthly visits, 14% received visits every other month and 22.95% received occasional visits.

Comparative Analysis of the Effect of the VaccinesTALOVAC 109 SE and SG VACCINE NOBILISin the Formation of Immunity Against Salmonella Enteritidis

Two groups of 60-day-old chickens of Hisex white breed were respectively vaccinated with inactivated TALOVAC 109 SE and live SG VACCINE NOBILIS. Four months after vaccination, blood sera tests of both experimental groups were performed using the enzime immunoassay(ELISA) and reactions of double diffusion in agar gel with antigens specific for salmonellae. By ELISA there were defined relative values S/N of antibodies against Salmonella enteritidis as well as titre means in both groups. Experimental data show that in the first group vaccinated with TALOVAC 109 SE the mean of S/N values was 0,47 of relative units, whereas in the second group vaccinated with SG VACCINE NOBILIS S/N mean was lower and equaled 0,54. Applying double diffusion in agar gel method, a common precipitation link was found in both groups. It indicates, that the antigen against which antibodies developed in the organisms of chickens is common for S. enteritidis and S. gallinarum pathogens.

A rapid and sensitive screening kit for the detection of anti- Toxocara larval ES antibodies

We developed a new screening kit for the detection of anti- Toxocara larval excretory-secretory antibodies. The test can be performed within 3 min for one sample and does not require a high-priced supplemental instrument. Moreover, results are easily and directly observed. Using this test kit, 22 sera taken from healthy subjects were negative for anti- Toxocara larval excretory-secretory antibodies at the serum dilution of 1:20. Of 14 proven cases of parasitic infections other than toxocariasis, one (gnathostomiasis) showed a positive result, but the others were negative. In serologically diagnosed toxocariasis, the test kit showed good correlation with ELISA, immunoblot and double gel diffusion tests. We designated this test kit as ToxocaraCHEK.

Uso de serologia na identificação de predadores de Orphulella punctata (De Geer) (Orthoptera: Acrididae)

Serological techniques were used to study Orphulella punctata (De Geer) predators. For preparation of antiserum, rabbits were immunized by the limphonodule injection method with antigens obtained from O. punctata male. Serological tests were performed using double diffusion in agar gel. Specific serological reactions were obtained 13 days after the 1st inoculation. After the 2nd inoculation 5 precipitation lines were observed. In the reactions with female antigens there were 2-4 lines, while egg antigens showed 2 lines. These variations in the number of lines were attributed to different physiological states and to ontogeny. Positive reactions were observed for spiders and chrysopids captured in the field.

Site-specific mutagenesis of Clostridium perfringens alpha-toxin: replacement of Asp-56, Asp-130, or Glu-152 causes loss of enzymatic and hemolytic activities.

The current study has investigated the role of D-56, D-130, and E-152 in zinc ion binding properties, as well as the hemolytic, phospholipase C (PLC), and sphingomyelinase (SMase) activities of Clostridium perfringens alpha-toxin, based upon crystallography studies of the Bacillus cereus PLC, which had suggested these residues might be important for these functional activities. The replacement of D-56 in alpha-toxin resulted in complete loss of hemolytic, PLC, and SMase activities. The variant toxins at D-130 showed an approximately 100-fold reduction of biological activities compared to that of the wild-type toxin. The substitution of glutamine or glycine for E-152 caused complete loss of these activities, but substitution of aspartic acid for E-152 reduced but did not completely inhibit these activities. The variant toxins at D-56 and D-130, as well as the wild-type toxin, possessed approximately 2 mol of zinc atoms per mol of the protein, but E152G and E152Q contained approximately 1 mol of zinc metal per mol of the protein. On the other hand, the zinc content in E152D was calculated as about 1.4 mol in the toxin molecule. The replacement of D-56, D-130, or E-152 had no effect on binding to sheep erythrocytes and uptake of free zinc ion from the solution. The variant toxins at D-130 showed partial antigenic identity with the wild-type toxin on a double gel diffusion test. These observations suggest that D-56 in alpha-toxin is required for catalytic activity of alpha-toxin, D-130 is essential for maintenance of structure, and the carboxyl group of E-152 tightly ligands one zinc ion, which is essential for catalytic activity of the toxin.

Detection of two isotypically different antibodies produced by a murine hybridoma.

A hybridoma, F31P46B, secreting monoclonal antibodies (mAbs) comprised of mu and gamma heavy chains in association with a single kappa light chain, has been characterized. This hybridoma was prepared by fusing splenocytes, derived from a BALB/c mouse immunized with Vibrio vulnificus and SP2/O-Ag-14 mouse myeloma cells. The specificity of this hybridoma was determined by ELISA screening on a large number of bacterial strains. Hybridoma cells of F31P46B were cloned by limiting dilution to an average cell density of 0.1 cells/well and repeated 3 times to ensure monoclonality. Isotyping of 7 subclones was then performed by Ouchterlony gel double diffusion, as well as a Bio-Rad isotyping kit, and both methods showed that both IgM and IgG2b were secreted. PAGE and immunoblotting showed the presence of mu, gamma, and kappa chains with respective molecular weights of 80, 50, and 25kDa. A series of fractions, collected from F31P46B ascites during Superose 12 gel chromatography, were tested by the two isotyping methods and each confirmed the presence of two immunoglobulin products. These data indicated that the hybridoma secreted two separate immunoglobulins, IgM/kappa and IgG2b/kappa.

Significance of Aspergillus fumigatus rDNA detected by polymerase chain reaction in diagnosis of pulmonary aspergillosis

We have studied the clinical significance of Aspergillus fumigatus rDNA detected by polymerase chain reaction (PCR) for diagnosing aspergillosis. For this purpose, a specific and sensitive PCR assay was developed to amplify the 26S rDNA/intergenic spacer region of A. fumigatus. Control experiments showed that the set of primers used was capable of amplifying A. fumigatus DNA specifically and that the DNA amount of detection limit was 1 pg. Eighteen samples from 13 patients with aspergillosis and 36 samples from 24 patients without aspergillosis were tested by means of PCR, culture, latex agglutination test for galactomannan antigen and double gel diffusion assay for precipitation of antibodies to A. fumigatus. PCR showed positive test in 8 among 13 patients with pulmonary aspergillosis. On the other hand, culture of samples detected A. fumigatus in 6 patients. Galactomannan antigen and antibodies specific to A. fumigatus were positive in 2 and 6 patients respectively. These results indicated that PCR is the most sensitive among these 4 methods. Five patients showed negative PCR test despite of having pulmonary aspergillosis. Two of these patients were proved to have aspergillosis caused by A. flavus and A. niger. On the other hand, galactomannan antigen and A. fumigatus antibody were positive in one and 2 patients, respectively. PCR was positive in 2 out of 24 patients diagnosed as not having aspergillosis: one patient had diagnosis of acute bronchitis, but she showed positive culture of A. fumigatus. The other patient had diagnosis of lobar pneumonia, because any pathogens were not detected before PCR assay. The PCR assay we developed is a useful method for diagnosing aspergillosis caused by A. fumigatus as compared with other conventional methods.

Epidemiological aspects of group B streptococci of bovine and human origin.

Restriction fragment length polymorphism of the gene encoding rRNA (ribotyping) was used in combination with conventional epidemiological markers to study phenotypic variations among Streptococcus agalactiae of bovine origin and the possible epidemiological interrelationship between the bovine and human reservoirs of Streptococcus agalactiae. The bovine material constituted 53 strains (9 antigen combinations) isolated from 11 herds. Herds with a uniform as well as heterogenic antigenic pattern were included. Furthermore, strains isolated in the course of time from the same persistently infected quarters were examined. The human material constituted 16 strains, 4 each of 4 serotypes, isolated from healthy carriers. Finally, nine serotype- and the group reference strains were examined. All strains were serotyped by double diffusion in agarose gel, biotyped (lactose +/-), and ribotyped using two restriction enzymes, Hind III and HhaI. All isolates could be typed by ribotyping and seven ribotypes were identified among the reference strains. The restriction enzymes used alone or in combination gave typing results that allowed discrimination between and within serotype. Combined use of serotype, Hind III and HhaI ribotypes produced 11 types among the 16 human strains. Ribotype analysis discriminated between herds infected with the same serotype. Strains of varying antigenic patterns from the same herd had the same ribotype. Phenotypic variations in serotype observed in persistent intramammary infection were not related to genetic changes as monitored by ribotype. Two ribotypes were represented among both bovine and human strains. The discriminating capability of lactose fermentation was of limited value.

Structural and serological studies of lipopolysaccharides of Citrobacter O35 and O38 antigenically related to Salmonella.

Structural analysis using 13C NMR spectroscopy and methylation showed that lipopolysaccharides (LPSs) of Citrobacter freundii O35 and Salmonella arizonae O59 have structurally identical O-specific polysaccharide chains, and those of C. freundii O38 and Salmonella kentucky differ only in the presence of O-acetyl groups in the former. Serological relationships between the structurally similar LPSs were demonstrated using inhibition of ELISA, rocket immunoelectrophoresis, double gel diffusion, and immunoblotting. The O-acetyl groups present in C. freundii O38 LPS are of little importance for its serological specificity. A cross-reaction was observed in immunoblotting between O-antisera to C. freundii O35 and S. arizonae O59 and a structurally related LPS of Pseudomonas aeruginosa O11a, 11b (Lányi-Bergan classification).

Structural and serological studies of lipopolysaccharides of Citrobacter O35 and O38 antigenically related to Salmonella

Structural analysis using 13C NMR spectroscopy and methylation showed that lipopolysaccharides (LPSs) of Citrobacter freundii O35 and Salmonella arizonae O59 have structurally identical O-specific polysaccharide chains, and those of C. freundii O38 and Salmonella kentucky differ only in the presence of O-acetyl groups in the former. Serological relationships between the structurally similar LPSs were demonstrated using inhibition of ELISA, rocket immunoelectrophoresis, double gel diffusion, and immunoblotting. The O-acetyl groups present in C. freundii O38 LPS are of little importance for its serological specificity. A cross-reaction was observed in immunoblotting between O-antisera to C. freundii O35 and S. arizonae O59 and a structurally related LPS of Pseudomonas aeruginosa O11a,11b (Lányi-Bergan classification).

Serovars of Erysipelothrix Strains Isolated from Pigs Affected with Erysipelas in Japan.

Serovars of 1,046 Erysipelothrix strains isolated from diseased pigs during a period from 1983 to 1993 in Japan were determined by an agar gel double-diffusion precipitation system using typing sera representing all the known serovars, 1 through 23 and type N, of 2 species of Erysipelothrix. A total of 943 strains could be serotyped within the serovars. The remaining 103 strains could not be determined for their serovars and were classified as untypable. Of the 938 (99.5%) strains serologically identified as Erysipelothrix rhusiopathiae, 423 (40.4%) belonged to serovar 1a, 359 (34.3%) to serovar 2, 65 (6.2%) to serovar 1b, 35 (3.3%) to serovar 6, 19 (1.8%) to serovar 5, 11 (1.1%) to serovar 21, and the remaining 26 to serovars 4, 8, 11, 12, 15, 17, or 19. Only 5 (0.5%) strains isolated from the cases of erysipelas belonged to Erysipelothrix tonsillarum and represented serovars 7 or 23.

Evaluation of agar gel double diffusion test for detection of nuclear polyhedrosis virus of silkworm, Bombyx mori L.

The agar gel double diffusion test was evaluated for detection of nuclear polyhedrosis virus infecting the silkworm, Bombyx mori L. (BmNPV). The test was successful using alkali-dissolved BmNPV as antigen. The antiserum raised against dissolved BmNPV gave positive results with a wider range of antigenic concentrations compared to antiserum raised against undissolved BmNPV. Gel plates prepared using phosphate buffered-saline as dissolving medium for agarose gave sharper and clearer precipitation bands compared to those made with sodium carbonate-saline as diluent. Sharp bands were obtained when the experiment was carried out at 25°C, whereas at higher temperature (35°C), the bands were diffused. The test was able to detect antigen concentration as low as 2 × 106 OBs/ml (18.9 μg/ml of antigen protein).

Serological relationships among rice yellow mottle virus isolates

SummarySerological studies on five isolates of RYMV collected from the Ivory Coast (IC), Sierra‐Leone (SL), Niger (Nr), Kenya (K) and Nigeria (N) indicated that these isolates are serologically related. Gel double diffusion and direct ELISA tests showed that the five isolates could be arranged into three serological groups here designated RYMV‐N, SL‐IC and K‐NR. However, the ISEM studies did not reveal any clear grouping of the heterologous isolates tested.

Construction of mutant genes for a non-toxic verotoxin 2 variant (VT2vp1) of Escherichia coli and characterization of purified mutant toxins.

The gene encoding a Verotoxin 2 variant, VTvp1, was mutated by oligonucleotide-directed site-specific mutagenesis. Among 6 mutant toxins encoded by the mutated genes, E167Q-R170L (glutamic acid at position 167 and arginine at position 170 from N-terminus of the A subunit were replaced by glutamine and leucine, respectively) was found to have markedly decreased activities; inhibition of protein synthesis, Vero cell cytotoxicity and mouse lethality of the purified E167Q-R170L were 1/1,900, 1/125,000 and 1/2,000, respectively, of those of the purified wild-type VT2vp1. Since the antigenic property of the E167Q-R170L was demonstrated to be similar to that of the wild-type VT2vp1 by Ouchterlony double gel diffusion test and by neutralization test of Vero cell cytotoxicity of the VT2vp1, a possibility to use the mutant VT2vp1, E167Q-R170L, as a toxoid is discussed.

Identification of a Strain of Peanut Chlorotic Streak Virus Causing Chlorotic Vein Banding Disease of Groundnut in India

AbstractA virus disease characterized by chlorotic vein banding, chlorotic line pattern along the margins or midrib of mature leaflets and chlorotic spots/rings was observed on commercial groundnut crops in Rayalaseema area of Andhra Pradesh with an incidence from 1% to nearly 60%. The virus was transmitted by mechanical inoculation in extracts prepared with 0.01 M potassium phosphate butter, pH 8.0 to 21 species from the Chenopodiaceae, Cruciferae, Leguminosae and Solanaceae, Chenopodium quinoa was found to be a good local lesion host. The virus was neither seed‐transmitted through 1591 groundnut seeds nor aphid‐transmitted by Aphis craccivora, Myzus persicae and Rhopalosiphum maidis either in non‐persistent or semi‐persistent manner. The virus remained infective in buffered tobacco leaf sap at a dilution of 10−5; in a 10−1 dilution of buffered sap the virus was infective for 2–3 days at 22–29°C or when heated to 65°C for 10 min but not to 70°C.Clarification treatments with organic solvents with 10% chloroform was least damaging. The virus was purified from Nicotiana rustica leaves. Purified virus contained isometric particles of 51 nm in diameter with an electron dense core of 22 nm and two major polypeptides of 76 kDa and 36 kDa. A polyclonal antiserum to this virus was produced. In agar gel double diffusion, enzyme‐linked immunosorbent assay and in electro‐blot immunoassay rests the virus was related to peanut chlorotic streak virus and not to cauliflower mosaic, figwort mosaic and soybean chlorotic mottle viruses.

沈降抗体検出用アスペルギルス粗抗原に関する若干の検討 補遺

We reported earlier on culture filtrate antigens from A. fumigatus that had been incubated at 25°C for 1-10 weeks (Jpn J Med Mycol 34: 439-444, 1993). Additionally, we prepared five culture filtrate antigens from A. fumigatus ATCC26430 grown by shaking at 25°C for 11-15 weeks in Sabouraud liquid medium. We also prepared a polysaccharide antigen from the same strain by extracting mycelia and conidia. The growth of the fungi became weaker in cultures after 12 weeks, and by stopped at 14 weeks. Sixteen antigens were then employed in agar gel double diffusion (DD) tests against the sera of 18 aspergilloma patients. The results of DD showed three types (A, B, C) of precipitin pattern, with 9 patients belonging to type A, 6 to type B and 3 to type C. In all cases, precipitin lines were formed with the antigens obtained after 6-15 weeks of culture. To avoid contamination, inferior cultures and accumulation of waste, the incubation time should be as short as possible. Thus, as noted in our previous letter, we found that the antigen obtained from 6-week culture filtrates at 25°C is the most useful for demonstrating precipitin reactions against the sera of aspergilloma patients.

Easy-to-perform modified Elek test to identify Shiga-like toxin-producing diarrhoeogenic Escherichia coli

We determined whether Shiga-like toxin I (SLT-I) -producing diarrhoeogenic Escherichia coli could be detected by a modified Elek test. The test (SLT Elek test) is based on the principle of the Elek test and the Ouchterlony double-gel diffusion. The development of the SLT Elek test was preceded by a preliminary study; the purpose of the latter was to establish whether a simplified purification procedure of SLT-I (involving bacterial sonic extract, “Affi-Gel Blue” chromatography and anion- and cation-exchange liquid chromatography) could be employed in the preparation of rabbit antisera to SLT-I. SLT-I-specific antisera were obtained after adsorption of sera with bacterial sonic extract from non-toxigenic E. coli. A total of 135 strains of E. coli were tested by the SLT Elek test (100 SLT-I-negative and 35 SLT-I-positive). The results of the SLT Elek test and the Vero cell test correlated well: 30 strains gave positive results and 100 strains gave negative results in both tests. Only 5 strains gave discrepant results: they were weakly positive in the Vero cell assay, whereas 3 gave borderline reactions and 2 were negative in the SLT Elek test. Positive predictive value was 1, negative predictive value was 0.98; the SLT Elek test was 91 % sensitive and 100 % specific.