Gel Double Diffusion Tests Research Articles

Genetic analysis of an Erysipelothrix rhusiopathiae swine isolate determined to be serovar 2 by a gel double diffusion test but serovar 1a/2 by a serotyping PCR assay.

We previously developed a multiplex PCR assay for the differentiation of serovar 1a, 1b, 2 and 5 strains of Erysipelothrix rhusiopathiae. In this study, we analyzed the serovar-defining chromosomal region of a serovar 2 swine isolate, which was PCR-positive for both serovars 1a and 2 by the multiplex PCR assay. Genetic analysis of the chromosomal region revealed that, as in serovar 1a strains, the ERH_1440 gene, which is usually truncated or missing in serovar 2 strains, was intact in this strain. This paper first shows an E. rhusiopathiae serovar 2 strain possessing an intact ERH_1440 gene and suggests that care may be needed when determining the serovar of such rare strains by PCR assay.

Development of an immunochromatographic strip for detection of antibodies against duck Tembusu virus

Duck Tembusu virus (DTMUV), a novel flavivirus, causes severe disease in ducks. There is an urgent need for a rapid and effective diagnostic method to control the spread of DTMUV. We chose the envelope (E) protein from DTMUV as an antigen and combined it with colloidal gold particles as tracers to specifically detect anti-DTMUV antibodies. Based on the double-antigen sandwich format, an immunochromatographic strip (ICS) for the rapid detection of anti-DTMUV antibodies was developed. The ICS showed a high specificity and no cross-reactivity with other sera. By detecting a serially diluted duck anti-DTMUV serum, the sensitivity of the ICS was 16-fold higher than that of the agar gel double diffusion test. Moreover, the ICS was both stable and reproducible, maintaining the same performance at 4°C for at least 6 months. To evaluate the effectiveness of the ICS, 217 duck serum samples were tested with the ICS and an indirect enzyme-linked immunosorbent assay (iELISA). The consistency ratio of positive and negative results between the two methods was 97.87% and 97.06%, respectively. The agreement between the ICS and the ELISA was 97.24%. The ICS developed in this study offers a specific, sensitive, and rapid method to detect anti-DTMUV antibodies.

Campylobacter hyointestinalis Isolated from Pigs Produces Multiple Variants of Biologically Active Cytolethal Distending Toxin.

Campylobacter hyointestinalis isolated from swine with proliferative enteritis often is considered to be pathogenic. While the precise virulence mechanisms of this species remain unclear, we have recently identified a cytolethal distending toxin (cdt) gene cluster in C. hyointestinalis isolated from a patient with diarrhea (W. Samosornsuk et al., J Med Microbiol, 27 July 2015, http://dx.doi.org/10.1099/jmm.0.000145). However, the sequences of the cdt genes in C. hyointestinalis were found to be significantly different and the gene products are immunologically distinct from those of other Campylobacter species. In this study, we demonstrate the presence of a second variant of the cdt gene cluster in C. hyointestinalis, designated cdt-II, while the former is named cdt-I. Sequencing of the cdt-II gene cluster and deduced amino acid sequences revealed that homologies between the subunits CdtA, CdtB, and CdtC of ChCDT-I and ChCDT-II are 25.0, 56.0, and 24.8%, respectively. Furthermore, the CdtB subunit of ChCDT-II was found to be immunologically unrelated to that of ChCDT-I by Ouchterlony double gel diffusion test. Recombinant ChCDT-II also induced cell distention and death of HeLa cells by blocking the cell cycle at G2/M phase. Interestingly, the cdt-II genes were detected in all 23 animal isolates and in 1 human isolate of C. hyointestinalis, and 21 of these strains carried both cdt-I and cdt-II gene clusters. Altogether, our results indicate that ChCDT-II is an important virulence factor of C. hyointestinalis in animals.

Identification of some viruses causing mosaic on lettuce and characterization of Lettuce mosaic virus from Tehran Province in Iran

Lettuce mosaic virus (LMV), Cucumber mosaic virus (CMV) and Tomato spotted wilt virus (TSWV) were identified in lettuce fields in Tehran province. In this study, 452 infected lettuce plants having viral infection symptoms including, mosaic, mottling, leaf distortion, stunting defective heading, were collected from the fields throughout Tehran province. Distribution of Lettuce mosaic virus (LMV), Cucumber mosaic virus(CMV), Tomato spotted wilt virus (TSWV) and Arabis mosaic virus (ArMV) were determined with DAS-ELISA. LMV, CMV and TSWV were found on lettuce in this region, but no infection by ArMV was found. Percentage of single infection by LMV, CMV or TSWV was 21, 16 and 10% respectively. Also, 16% of samples were co-infected with LMV+CMV, 8% with LMV+TSWV and 8% with CMV+TSWV. 5% of samples were infected to all of these viruses. LMV was found in 49%, CMV in 44% and TSWV in 31% of samples totally. Therefore, LMV is major agent of lettuce mosaic disease in Tehran province. This is the first report of occurrence of TSWV on lettuce in Iran and the first report of CMV and LMV in Tehran province. Three LMV infected samples were collected and their characteristics were determined. After mechanical inoculation, these isolates produced symptoms on Chenopodium quinoa, Chenopodium amaranticolor, Gomphrena globosa, Nicotiana benthamiana, Lactuca sativa cv. Mantilia and cv. Terocadero (which contains the mo11 resistance gene and susceptible respectively), but not cv. Salinas 88 (which contains the mo12 resistance gene). LMV was purified and LMV polyclonal antiserum was produced in rabbit by a series of intravenous and intramuscular injections, the titre of this antiserum was 1:1024. Gel double diffusion test (GDDT) was performed, and precipitin bands appeared. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting showed the presence of coat protein 29 kDa. In Immunocapture reverse transcription PCR (IC-RT-PCR) with a LMV specific primer pair, an aproximately 1300 bp fragment was amplified. Key words: Lettuce mosaic virus, Cucumber mosaic virus, Tomato spotted wilt virus, Immunocapture reverse transcription PCR, sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Microscopic and polyclonal antibody-based detection of yellow leaf disease of arecanut (Areca catechu L.)

Phytoplasma, the pathogen of yellow leaf disease (YLD) of arecanut (Areca catechu L.) was detected by transmission and scanning electron microscopy. Tissues of YLD affected palms contained phytoplasmas in the phloem sieve elements, but not in symptomless healthy palm tissues. Phytoplasma was purified from tissues of diseased palms employing percoll density gradient centrifugation and confirmed by transmission electron microscopy. Using the purified phytoplasma preparation, a polyclonal antiserum was raised in rabbits and used for standardisation of agar gel double diffusion (Ouchterlony) test and DAC-ELISA. Clear precipitin line was observed in Ouchterlony test between the antigen from diseased palms and the pathogen-specific antibodies after 48 h incubation and only undiluted antiserum showed best result in the test. However, in ELISA, 1:10 antigen dilution and 1:400 pathogen-specific antibodies dilution produced sensitive detection of the pathogen with a difference of >3.5 times absorption values between healthy and diseased samples. The results thus confirmed the ability of antiserum to distinguish healthy and infected plants and utility of ELISA for effective diagnosis of YLD.

Comparisons of complete RNA-2 sequences, pathological and serological features among three Japanese isolates of Arabis mosaic virus

Arabis mosaic virus lily and narcissus isolates (ArMV-Li and ArMV-Na) induced severe necrotic spots on Chenopodium quinoa, whereas ArMV butterbur isolate (ArMV-Bu) caused symptomless infection in the plant. The accumulation level of ArMV-Bu in upper non-inoculated leaves of C. quinoa was comparable to that of ArMV-Li or -Na. The agar gel double-diffusion test using an antiserum against ArMV-Li showed ArMV-Li was closely related to ArMV-Na, but not to ArMV-Bu. The RNAs-2 of ArMV-Li, -Na, and -Bu consist of 3707, 3709, and 3789 nucleotides, and they contain one open reading frame encoding a putative polyprotein of 1083, 1084, and 1122 amino acids, respectively. The overall identity of RNA-2 of ArMV-Li displayed more than 90% with ArMV-Na, but less than 70% with ArMV-Bu. A phylogenetic analysis of 2A sequences from ArMV isolates revealed ArMV-Bu was not categorized in any cluster. ArMV-Bu is a unique isolate from the point of view of pathological and serological features, and nucleotide sequence.

Natural Occurrence of Cucumber mosaic virus on Rauvolfia serpentina, A New Record.

Rauvolfia serpentina, family Apocynaceae, is widely cultivated in India and adjoining countries for the production of roots used in several herbal formulations (1). Severe mosaic and stunting of the whole plant was observed on R. serpentina growing in experimental plots of NBRI, Lucknow, in 2006. The causal pathogen was transmitted by sap inoculation on Nicotiana tabacum cv. White Burley, N. rustica, and N. glutinosa, which produced necrotic local lesions and systemic mosaic. The virus reacted positively with antiserum of Cucumber mosaic virus (CMV; PVAS 242a, ATCC, Manassas, VA) in gel double diffusion tests, indicating the presence of CMV. Total RNA was isolated from infected and healthy leaf tissues of R. serpentina, and reverse transcription (RT)-PCR was performed using CMV specific primers AM180922/AM180923. RT-PCR resulted in an expected size (~650 bp) amplicon in infected but not healthy samples. The amplicon was cloned, sequenced, and the data was submitted to GenBank (Accession No. DQ914877). BLAST search analysis of the virus isolate showed highest (99%) sequence identity with CMV isolates (GenBank Accession Nos. DQ640743, AF350450, X89652, and AF281864). The virus isolate showed a close phylogenetic relationship with Indian isolates of CMV belonging to subgroup IB. A literature survey revealed that there is no report of occurrence of any virus on R. serpentina except some fungal infections (2). To our knowledge, this is the first record of natural occurrence of CMV on R. serpentina.

동부(Vigna sinensis)의 국부병반을 이용한 Cucumber mosaic virus와 Broad bean wilt virus 2의 구별

Cucumber mosaic virus(CMV) and Broad bean wilt virus 2(BBWV2) were isolated from Gentiana scabra plants showing typical mosaic and yellowing symptoms. When the inoculum of mosaic symptom propagated in Nicotiana benthamiana was inoculated to primary leaves of Vigna sinensis, the local lesions of different types was developed. Type one produced a small necrotic spot(SNS) of pinpoint type, while the other one showed a large necrotic spot(LNS) of halo type. LNS on primary leaves of V. sinensis was also induced by inoculum from yellowing symptom on G scabra. Single lesion from SNS induced a typical mosaic symptom on N. Benthamiana. On the other hand, LNS produced a chlorotic ring symptom on inoculated leaves and mosaic plus necrotic ringspot on upper leaves of N. benthamiana. An isolate of CMV from SNS and BBWV2 from LNS were detected by using dsRNA analysis, RT-PCR and agar gel double-diffusion test. Thus, our results should provide a tool of a simple method for discrimination from mixed infected plants by CMV and BBWV2.

Pepper mild mottle virus에 대한 난황항체의 생산과 혈청학적 진단에의 활용

Egg yolk immunoglobulin (IgY) is much widely used in medical fields, but its use in serology of plant viruses is much limited. We produced an IgY against pepper mild mottle virus (PMMoV) and applied it to several serological tests. Polyclonal antibodies were obtained from the egg yolk of chicken immunized with a total of 2mg of purified PMMoV over 2 months. The titers of antibodies were measured with the ring-test over six months after the first injection. The highest.titers of IgY was 1/2,560 at 2 months after the first injection. Approximately 60-80 mg of IgY were obtained from one egg yolk. Using the IgY, 1ng/ml of purified PMMoV was detected with the indirect ELISA. Gelrite gel double diffusion test, ELISA and tissue immuno-binding assay employing IgY gave similar sensitivity and specificity to those of IgG developed in rabbit. Therefore, the IgY which can be obtained in large quantities from a chicken, might be useful for the antibody production and the serology of plant viruses.

Production and properties of monoclonal antibodies to Solanum nodiflorum mottle sobemovirus

SummaryBlack raspberry necrosis virus (BRNV) reaches only very low concentrations in herbaceous plants and is difficult to maintain in culture. However, in a mixed culture with an unrelated virus, Solanum nodiflorum mottle (SNMV), in the genus Sobemovirus, the concentration of BRNV particles increases about 1000‐fold. In attempts to produce monoclonal antibodies (MAbs) to BRNV for diagnostic use, purified virus particles from the mixed virus culture were used as immunogen and the resultant antibodies screened against cultures of SNMV alone, BRNV+SNMV and healthy plant extracts. None of the virus‐specific MAbs obtained in this way was specific to BRNV but six were specific to SNMV. Although the original objective was not achieved, the SNMV MAbs were characterised and used to study serological properties of SNMV and other Sobemoviruses. Characterisation of the six SNMV MAbs showed that four were IgG3, one IgG1 and the other IgG2b. SNMV was detected by all six MAbs in ELISA, by five in Western blotting, by three in agarose gel double diffusion tests, but only one was suitable for trapping virus particles in immuno‐electron microscopy (IEM). In Western blotting using virus in sap extracts of Nicotiana clevelandii, each of the five MAbs detected a single major band of Mc. 31 000 in sap containing SNMV, and additional bands of lower mass attributed to degradation of coat protein. In various serological tests, no cross‐reactions were detected between SNMV and seven other viruses from the genus Sobemovirus. However, in IEM but not in Western blotting, significant cross‐reactions were observed between SNMV and Velvet tobacco mottle virus, another species from the genus Sobemovirus. The significance of these different findings is discussed.

Identification of Tomato mosaic virus Infection in Lisianthus in Taiwan

In recent years, Lisianthus (Eustoma russellianum (Don.) Griseb) has become popular as potted plants and cut flowers in Taiwan. They are grown in the central and southern regions of the island. Since 1998, diseased plants with mosaic symptoms, followed by necrosis of leaf tissues, were observed in commercial greenhouses and field-grown lisianthus. Newly emergent leaves were curled and smaller compared with those on healthy plants. These symptoms greatly decreased the commercial value of the crop. Rigid rods similar to tobamoviruses that measured 300 × 18 nm were found consistently associated with symptomatic plants. In July 2002, a virus culture was isolated from diseased lisianthus from Chiayi County, Taiwan and established and maintained in systemic hosts Nicotiana tabacum L. and N. benthamiana Domin. Chlorotic and necrotic spots developed on lisianthus leaves 1 to 2 weeks after inoculation with the virus; symptoms eventually became systemic. Virions were purified from inoculated N. tabacum. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that the virus contained one 18-kDa (Mr) polypeptide. The virus reacted positively in agar gel double diffusion tests and enzyme-linked immunosorbent assays with antisera prepared to Tobacco mosaic virus (TMV) or Tomato mosaic virus (ToMV) (gifts of S. D. Yeh, National Chung Hsing University, Taichung, Taiwan). A viral coat protein (CP) gene approximately 0.5 kb was amplified by reverse transcription-polymerase chain reaction from total RNA prepared from infected N. benthamiana using 5'-GCGAGCCATGGATTCTTACTCAATTACT as a forward primer and 5'-ACTCTCGGATCCTTAAGATGCAGGTGCAGA as a reverse primer. Comparison of the 480-nt CP gene region with that of ToMV-OM (GenBank Accession No. X02144) (3) revealed 99.2% nucleotide identity and 99.4% amino acid identity. It shares, however, 74.4% nucleotide identity and 83.9% amino acid identity with CP genes of TMV-U1 (GenBank Accession No. AX040174) and TMV-vulgare (GenBank Accession No. J02415) (1). The virus induced local lesion responses similar to ToMV on inoculated N. tabacum cv. White Burley, N. sylvestris Speg. & Comes, and Datura stramonium L. Inoculation of TMV, however, resulted in a systemic infection in these plants. Results from sequence analysis and diagnosis based on host reaction to the virus inoculation indicated that the tobamovirus infecting lisianthus in Taiwan is an isolate of ToMV. The virus is economically important to lisianthus and tomato in Taiwan. To our knowledge, this is the first report of ToMV causing disease on lisianthus in Taiwan. The disease was previously observed on lisianthus in Italy (2).

GRAPEVINE DEFORMATION VIRUS, A NOVEL NEPOVIRUS FROM TURKEY

A virus with isometric particles ca. 30 nm in diameter and angular contour was recovered by mechanical transmission from a grapevine with fanleaf-like symptoms growing at Nevsheir (Cappadocia, Turkey). In sucrose density gradient centrifugation this virus (laboratory code: isolate N66) sedimented as three components, T (empty shells), M, and B, both of which consisted of apparently intact particles. Virus preparations contained two RNA species with mol. wt. 2.6·106 Da (RNA-1) and 1.3·106 Da (RNA-2). The coat protein (CP) subunits were of a single type with Mr of c. 53,000. An antiserum with a titre of 1:1024 was raised, which did not react with healthy plant antigens. A fragment 1,175 nt in size from the 3’ terminal region of RNA-2 was sequenced, which comprised part of the CP cistron. Comparison analysis with GenBank sequences from the same region revealed variable levels of homology with other grapevine nepoviruses, the closest being Arabis mosaic virus (ArMV, 69% identity at the amino acid level) and Grapevine fanleaf virus (GFLV, 58% identity at the amino acid level), both belonging in subgroup A of the genus Nepovirus. Based on the determined sequence, specific primers were designed, which in RT-PCR assays amplified a 240 bp fragment from grapevine crude tissue extracts. The physicochemical properties of isolate N66 and the cytopathology of infected Chenopodium amaranticolor tissues resembled very much those of nepoviruses. In gel double diffusion tests isolate N66 proved serologically unrelated to 16 different nepoviruses. A distant positive reaction was obtained with ArMV in immunodiffusion (serological differentiation index = 4) and immunoelectron microscopy tests and when leaf extracts from infected grapevines or C. quinoa were tested in ELISA with commercial antisera to ArMV. These results support the notion that isolate N66 is a hitherto undescribed virus species belonging in subgroup A of the genus Nepovirus, serologically related to but distinct from ArMV, for which the name Grapevine deformation virus (GDefV) is proposed.

Genetic and immunological analysis of a novel variant of Shiga toxin 1 from bovine Escherichia coli strains and development of bead-ELISA to detect the variant toxin.

A novel variant of Shiga toxin 1 (Stx1) was identified from bovine Escherichia coli strains. The stx1 variant genes designated as stx1v51 and stx1v52 were cloned and sequenced. The two variant genes differed each other by 2 bp, but the deduced amino acid sequences of the two Stx1 variant toxins were the same and had 94% and 92% homology to that of prototype A and B subunits of Stx1, respectively. The variant toxin designated as Stx1v52 was purified to homogeneity. Although inhibition of protein synthesis in vitro by purified Stx1v52 was almost equal to that of purified Stx1, Vero cell cytotoxicity and mouse lethality of Stx1v52 were several folds lower than those of prototype Stx1. In Ouchterlony double gel diffusion test, the precipitin line between Stx1v52 and Stx1 formed a spur against anti-Stx1 serum but was fused against anti-Stx1v52 serum. Stx1v52 and Stx1v52-specific-bead-ELISA was developed, and both Stx1 and Stx1v52 could be detected with high sensitivity using Stx1v52 conjugate. However, Stx1v52 but not Stx1 could be detected with Stx1v52-specific bead-ELISA.

Elderberry latent virus: its relationship to Pelargonium ringspot virus and its identification as a distinct member of the genus Carmovirus, family Tombusviridae

Summary The properties of Elderberry latent virus (ELV) and Pelargonium ringspot virus (PelRSV) were compared. The viruses were largely indistinguishable in herbaceous host range and symptomatology, particle morphology, sedimentation coefficient and RNA profiles and size. They were also very closely related serologically with SDI differences in agarose gel double-diffusion tests of 1 to 3. Purified virus particle preparations of each virus contained isometric particles c. 30 nm in diameter that sedimented as a major component with an sO20W of 112–115S. Purified virus particle preparations contained a major and a minor ssRNA species that in polyacrylamide gel electrophoresis (PAGE) had estimated sizes of c. 3.8 kb and c. 1.6 kb respectively. Plants of Chenopodium quinoa infected with ELV or PelRSV each contained three dsRNA species of c. 3.8, 2.6 and 1.8 kbp, although the smallest of these species was not evident in all preparations. Protein from purified virus particle preparations contained a major polypeptide that, in SDS-PAGE, had an estimated Mr of 40 000 (40K). However, after storage of purified virus particles for 7–10 days, protein preparations from PelRSV particles also contained an additional major polypeptide of estimated Mr of 37 000 that is probably derived by degradation of the 40K protein; this additional component was not observed in freshly prepared preparations of ELV. Neither virus was found to be related serologically to 16 other viruses with isometric particles and similar properties. These data, together with the recent finding by other researchers that the smallest RNA species is a sub-genomic RNA, suggests that both viruses are members of the genus Carmovirus, and that PelRSV is a minor variant of ELV. However, the taxonomic status of these two viruses is discussed in relation to recent brief reports comparing the nucleotide and amino acid sequences of these two viruses.

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.

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.

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.

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.