Protective effects of clostridium sordellii LT and HT toxoids against challenge with spores in guinea pigs.

The protective effects of Clostridium sordellii lethal toxin (LT) and hemorrhagic toxin (HT) toxoids against challenge with spores in guinea pigs were investigated. Purified LT and partially purified HT were obtained from the culture supernatant of C. sordellii strain 3703, and then were treated with formalin to make toxoids. LT. HT and combined LT and HT (LT/HT) toxoid vaccines were prepared by mixing each toxoid with an aluminum phosphate gel as adjuvant. Guinea pigs immunized twice with the respective toxoid vaccines were challenged with spores of strains 3703 or KZ1047. The latter strain does not produce HT. LT toxoid vaccine conferred protection against challenge with strain KZ1047, but not strain 3703, in guinea pigs. All guinea pigs immunized with HT toxoid vaccine died after challenge with spores of either strain. LT/HT toxoid vaccine gave complete protection against challenge with spores of strains 3703 and KZ1047 to guinea pigs. These results suggest that not only LT toxoid, but also HT toxoid, are essential protective antigens of C. sordellii.

Amplification of the 16S-23S rDNA spacer region for rapid detection of Clostridium chauvoei and Clostridium septicum.

Amplification of the 16S-23S rDNA spacer region by polymerase chain reaction (PCR) was used for the rapid detection of Clostridium chauvoei and C septicum. To assess its specificity, PCR was performed with total DNA from 42 strains of clostridia and three strains of other genera. PCR products specific to C chauvoei or to C septicum were generated from homologous cultures only. Clostridium chauvoer-specific or C septicum-specific amplicons were also generated from tissues of cows experimentally infected with C chauvoei or C septicum and in DNA samples from cows clinically diagnosed as having blackleg or malignant oedema. These results suggest that a species-specific PCR may be useful for the rapid and direct detection of C chauvoei and C septicum in clinical specimens.

Quantitative diversity of 67 kda protective antigen among serovar 2 strains of Erysipelothrix rhusiopathiae and its implication in protective immune response.

Mouse monoclonal antibodies (MAbs), raised against the NaOH-extracted antigen of Erysipelothrix rhusiopathiae strain Kyoto (serovar 2), recognized two different epitopes on a single protein of molecular weight 67 kDa. The MAbs were classified as protective or non-protective against strain Fujisawa (serovar 1). In immunoblotting analysis using the MAbs, fifteen wild strains were shown to contain different amounts of 67 kDa protective antigen. Each formalin-killed whole cell vaccine (bacterin) prepared from the fifteen wild strains conferred different levels of protection against strain Fujisawa in mice. Bacterins prepared from wild strains with larger amounts of 67 kDa protective antigen tended to give high levels of antigen-specific antibody and better protection to mice. These results indicate that the amount of 67 kDa protective antigen which influences the induction of protective immune responses may vary substantially among the strains of E. rhusiopathiae (serovar 2).

Identification and characterization of Elongin A2, a new member of the Elongin family of transcription elongation factors, specifically expressed in the testis.

The Elongin complex stimulates the rate of transcription elongation by RNA polymerase II by suppressing the transient pausing of the polymerase at many sites along the DNA template. Elongin is composed of a transcriptionally active A subunit and two small regulatory B and C subunits, the latter of which bind stably to each other to form a binary complex that interacts with Elongin A and strongly induces its transcriptional activity. To further understand the roles of Elongin in transcriptional regulation, we attempted to identify Elongin-related proteins. Here, we report on the cloning, expression, and characterization of human Elongin A2, a novel transcription elongation factor that exhibited 47% identity and 61% similarity to Elongin A. Biochemical studies have shown that Elongin A2 stimulates the rate of transcription elongation by RNA polymerase II and is capable of forming a stable complex with Elongin BC. However, in contrast to Elongin A, its transcriptional activity is not activated by Elongin BC. Northern blot analysis revealed that Elongin A2 mRNA was specifically expressed in the testis, suggesting that Elongin A2 may regulate the transcription of testis-specific genes.

Structural organization and chromosome location of the mouse elongin A gene (Tceb3).

Elongin A is the transcriptionally active subunit of the Elongin complex, which strongly increases the rate of elongation by RNA polymerase II by suppressing the transient pausing of the polymerase at many sites within transcription units. In the present study, we obtained the cDNA sequence of the mouse Elongin A gene (Tceb3) and characterized its genomic structure. The deduced 773-amino acid sequence of mouse Elongin A shows 91% and 81% identity with rat and human Elongin A, respectively. The Elongin A gene was mapped to mouse chromosome 4D3 by fluorescence in situ hybridization.

Detection of porcine epidemic diarrhea virus using polymerase chain reaction and comparison of the nucleocapsid protein genes among strains of the virus.

Two pairs of PCR primers were designed to perform nested PCR targetting of a 540 bp fragment of the nucleocapsid (N) protein gene (N gene) of porcine epidemic diarrhea virus (PEDV). The N gene of PEDV was amplified with 4 PEDV strains and 11 small intestines of PEDV-infected piglets collected from 2 farms in Kagoshima prefecture, Japan. Nucleotide sequences of the PCR products from a Korean and two Japanese strains (KKN96-1 and S1) of PEDV isolated in 1993 and 1996, respectively, were almost identical. These results suggest that the PCR is an available tool for detection of PEDV from pigs in the field, and that the two Japanese strains (KKN96-1 and S1) were genetically similar to the Korean strain.

Characterization of flagellin from Clostridium chauvoei.

Differential centrifugation and cesium chloride-equilibrium centrifugation were used to purify the flagella from the strain Okinawa of the formalin-fixed Clostridium chauvoei. SDS-PAGE profile of purified flagella showed that a major protein band with a molecular mass of 46 kDa, corresponding to the flagellin monomer, and at least two minor protein bands with molecular masses of approximately 73 and 100 kDa were found. The amino acid composition of C. chauvoei flagellin was similar to the flagellin of Salmonella typhimurium and Bacillus subtilis. In addition, C. chauvoei flagellin monomer shared limited sequence homology with the N-terminal amino acid sequence reported for other bacterial flagellins. N-terminal sequences of two minor bands corresponded to the flagellin monomer, indicating that higher molecular mass bands were polymeric forms of the flagellin monomer.

Highly controlled heterologous gene expression through combined utilization of the tetracycline-repressible transactivator and the lac repressor.

To achieve strictly on-off switching of a desired cDNA expression in a broader range of mammalian cell types, we introduced the Escherichia coli lac operator sequences into the tetracycline-repressible promoter and created a chimeric promoter (designated here as the TcIP promoter) whose activity was reciprocally controlled by tetracycline and isopropyl thiogalactopyranoside (IPTG). cDNAs were connected downstream of the TcIP promoter and stably transfected into interleukin (IL-2 or IL-3)-dependent lymphoid cells that ectopically coexpress the tetracycline-repressible transactivator and the lac repressor. Whereas the parental tetracycline-repressible promoter exhibited constitutive activities when stably introduced into the lymphoid cells, cDNA expression from the TcIP promoter was strongly inhibited by tetracycline and was potently induced by IPTG in stable transfectants. Hence, the TcIP promoter made it possible to achieve highly controlled cDNA expression in cells wherein the parental promoter did not function in an inducible manner. Potential application of this promoter was provided by expressing cyclin-dependent kinase inhibitor, p27(Kip1). Induced expression of p27(Kip1) by the TcIP promoter in the lymphoid cells strongly reduced cellular responsiveness to IL-2 or IL-3, consistent with the idea that p27(Kip1) is a critical target that must be inactivated by the interleukin-triggered mitogenic signals.

Control of retinoblastoma protein-independent hematopoietic cell cycle by the pRB-related p130.

The retinoblastoma tumor suppressor protein (pRB) is a potent inhibitor of mammalian cell growth and the functional inactivation of pRB is widely presumed to be essential for progression of the cell cycle from G1 phase. In this work, the generality of pRB-based cell cycle control in mammalian cells was addressed by conditionally expressing pRB in cytokine-dependent hematopoietic cells. We show herein that these cells are able to progress through the cell cycle in response to cytokine despite the continued presence of supraphysiological amounts of wild-type pRB or phosphorylation-resistant pRB mutants. However, their growth was strongly blocked by ectopic expression of the pRB-related pocket protein, p130. This growth inhibition required the E2F-binding pocket domain but not the cyclin-binding domain of p130. Furthermore, increased amounts of the p130-controlled E2F, termed E2F-4, potentiated the mitogenic response of the cells to cytokine and the constitutive overexpression of E2F-4 rendered the cells cytokine-independent. Our results indicate the existence of a non-pRB-based cell cycle whose operation depends primarily on the interplay between p130 and E2F-4 in certain hematopoietic cells.

The protective effect of Clostridium novyi type B alpha-toxoid against challenge with spores in guinea pigs.

Clostridium novyi (C. novyi) Type B alpha-toxin was purified from culture supernatant by column chromatography, and was inactivated by formalin. A purified alpha-toxoid vaccine was prepared by mixing it with an aluminum phosphate gel adjuvant. Guinea pigs immunized twice with 4 micrograms or more of alpha-toxin survived against challenge with C. novyi Type B spores. Anti-alpha-toxin (antitoxin) titer was measured by toxin neutralization test using Vero cells. All of the guinea pigs having antitoxin titers of 10 units (U) or more at challenge were survived. In another experiment, guinea pigs were immunized with crude alpha-toxoid vaccines prepared by inactivated culture supernatant or by adding broken bacterial cells to the former. In this experiment, 10 U of antitoxin titer was the border of survival or death after challenge. Guinea pigs with antitoxin titers of less than 5 U, 5 U and 10 U died at 2, 3 to 4 and 4 days, respectively, after challenge. These results suggest that C. novyi alpha-toxin was the main protective antigen against challenge exposure to spores in guinea pigs.

Protective effect of NaOH-extracted Erysipelothrix rhusiopathiae vaccine in pigs.

A vaccine was prepared from a NaOH-extracted antigen of the Kyoto strain (serovar 2) of Erysipelothrix rhusiopathiae (E. rhusiopathiae) with an oil adjuvant, and was injected twice at 3-week intervals into SPF pigs and conventional pigs with maternal antibodies. After the second vaccination, IgG-GA titers of immunized SPF pigs were more than 256-fold at 3 weeks, and immunized pigs with maternal antibodies were 64-fold at 7 weeks. The pig with maternal antibodies vaccinated once with live vaccine had less than 4-fold titers. The ELISA antibody titers which were measured by using the NaOH-extracted antigen showed similar transition to the IgG-GA antibody titers. All immunized pigs and nonvaccinated control pigs were challenged with the strains Fujisawa (serovar 1a) or Saitama-1 (serovar 2). After challenge exposure, all pigs immunized with the NaOH-extracted vaccine showed no clinical signs and survived, and the pig immunized with the live vaccine had a local rhomboidal lesion at the site of the injection. Nonvaccinated pigs developed typical symptoms of E. rhusiopathiae infection and one of them died. After the autopsy, the challenge strains were not recovered from the main organs except tonsils of the pigs immunized with the NaOH-extracted vaccine. These results indicated that the NaOH-extracted vaccine induces a protective effect in pigs with maternal antibodies as well as in SPF pigs negative for such antibodies, and that 67-64, 62-60 kDa proteins in the NaOH-extracted antigen play an important role in protecting against E. rhusiopathiae infection.

A field trial of oil adjuvanten trivalent Actinobacillus pleuropneumoniae vaccine.

The trivalent vaccine of A. pleuropneumoniae serotype 1, 2 and 5 (AP3V) was prepared in the oil-in-water type adjuvanten form. At an SPF farm, the vaccinated pigs were observed for their antibody response, finishing rate, and lung lesions at the time of slaughter and for injection scars. The CF titers against serotype 1, 2 and 5 started to rise after the second injection, showed the highest titer at 30 days after injection and then gradually decreased in vaccinated pigs. The finishing rate in the vaccinated group was 91.6% and that in the control group immunized with commercial vaccine was 60%. The lungs in the control pigs showed severe pneumonia with hyperemia, pleural adhesion and abscess. In contrast, vaccinated pigs showed slight pneumonia. Injection scars were not observed in vaccinated pigs 100 days after the second injection. In conclusion, the pigs immunized with AP3V were sufficiently protected against A. pleuropneumoniae infection and the trial proved to be satisfactory in the safety of the vaccine under field conditions.

[In situ detection of Epstein-Barr virus in the non-neoplastic gastric glands and infiltrated lymphocytes].

Recently, clonal EBV-DNA and/or EBV-encoded small RNA(EBER1) have been detected in some gastric carcinomas. We reported the first observation of EBV infection in gastric glands with intestinal metaplasia, and characterized the EBV-infected lymphocytes which infiltrated in gastric mucosa. To determine the cellular location of EBV, EBER1 in situ hybridization(ISH) with an EBER1 oligonucleotide probe was applied to paraffin sections of the non-neoplastic gastric mucosa in 80 cases of gastric carcinoma and 49 cases of gastric ulcer. Not only was EBER1 expression detected in the nucleus of gastric cancer cells(5 cases) but also in non-neoplastic gastric epithelial cells(3 cases) and in infiltrated lymphocytes(40 cases). A single or a few shedding non-neoplastic epithelial cells in 2 cases of EBV-associated gastric cancer showed EBER1 expression. In one case, EBER1 was observed in all of the epithelial cells of a few gastric glands with intestinal metaplasia, suggesting that the stem cells of the metaplastic gastric glands were infected with EBV. EBV DNA was also detected in the EBER1-positive metaplastic glands scratched from the paraffin section by a single cell PCR method with a BamHI W primer pair. However, immunohistochemical examination showed that these metaplastic glands lacked expression of EBNA2 and LMP-1. The observation of these rare EBV-infected metaplastic glands reinforces the pre-transformation EBV infection hypothesis for EBV-associated gastric carcinoma. The double staining using ISH and immunohistochemistry revealed that EBER1 positive lymphocytes showed a B cell marker of L26(< 50%) but not T cell markers of UCHL1 and OPD4.