Pasteurella multocida- and Pasteurella haemolytica-ghosts: new vaccine candidates.

Pasteurella multocida is an important animal pathogen. Bacterial ghosts produced by the expression of phage PhiX174 lysis gene E are empty cells devoid of cytoplasmic and genomic material. Lysis of P. multocida 7A and P. haemolytica A1 carrying Pasteurella-specific lysis vectors (pSR2 and pSON2) occurred 140 min after induction of gene E expression induced by temperature upshift. The E-mediated cell lysis and killing activity was the same in both Pasteurella species and no viable cells could be detected after lysis of P. multocida and P. haemolytica. Pasteurella ghosts were used for immunization of rabbits and mice. Rabbits immunized subcutaneously with either P. multocida- or P. haemolytica-ghosts developed antibodies reacting with the immunizating strain, as well as with other Pasteurella strains. The number of proteins in whole cell protein extracts recognized by the sera constantly increased during the observation period of 51 days. In addition, dose-dependent protection against homologous challenge was observed in mice immunized with P. multocida-ghosts. Animals which received 1.15 x 10(8) ghosts and a challenge dose of up to 60 cfu (LD90), showed 100% protection. According to these results, we suggest ghosts of P. multocida and P. haemolytica as new vaccine candidates.

Protective immunity against pasteurellosis in cattle, induced by Pasteurella haemolytica ghosts.

Pasteurella haemolytica is a cattle pathogen of significant economic impact. An effective vaccine against bovine pneumonic pasteurellosis is therefore of high importance. Apart from economic concerns, pasteurellosis caused by P. haemolytica is a serious disease leading to death in cattle if it remains untreated. In this study P. haemolytica-ghosts are presented as a promising vaccine candidate in cattle. To obtain sufficient vaccination material a fermentation protocol for P. haemolytica-ghost production was established. With the obtained experimental P. haemolytica-ghost vaccine, cattle immunization studies were performed based on a Pasteurella cattle challenge model developed specifically for vaccine validation. It was shown that protective immunization of cattle against homologous challenge was induced by adjuvanted P. haemolytica-ghosts. The level of protection was similar to a commercially available vaccine.

Naturally occurring Clostridium perfringens nontoxic alpha-toxin variant as a potential vaccine candidate against alpha-toxin-associated diseases.

Clostridium perfringens mutant strain 121A/91 shows neither enzymatic (phospholipase C) nor hemolytic activity. Nevertheless, the cpa gene and the corresponding alpha-toxin variant are detectable. Vaccination with this genetically constructed alpha-toxin variant, rAT121/91, induces antibodies capable of significantly reducing activities induced by wild-type toxin. Thus, rAT121/91 could be a useful vaccine candidate.

Expression of a VEGF-like protein from Parapoxvirus ovis in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe.

We report on the expression of a VEGF-like protein encoded by Parapoxvirus ovis in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. We show that a lysine residue at amino acid position 2 (K2) is an important determinant for the stability of this protein in S. cerevisiae. Replacement of K2 by an arginine results in stabilization of the protein. This observation suggests that this lysine may be a target for ubiquitinylation, which is a prerequisite for proteasome-mediated protein degradation. Interestingly, in S. pombe the lysine (K2) has no influence on the stability of the protein. This result indicates that the two yeast species exhibit significant differences in their protein degradation pathways.

Quantitative detection of Chlamydia spp. by fluorescent PCR in the LightCycler.

Quantitative detection of intracellular bacteria of the genus Chlamydia by the standard cell culture method is cumbersome and operator dependent. As an alternative, we adapted hot-start PCR to the glass capillary quantitative PCR format of the LightCycler. The optimized PCR was consistently more efficient than commercially available pre-assembled PCRs. Detection by quantitative PCR of as few as single copies of DNA of Chlamydia spp. was accomplished by SYBR Green fluorescence of the dsDNA product and by fluorescence resonance energy transfer (FRET) hybridization probes. The PCRs were 15-fold more sensitive than the cell culture quantitative assay of C. psittaci B577 infectious stock. The number of chlamydial genomes detected by C. psittaci B577 FRET PCR correlated well with cell culture determination of inclusion forming units (IFUs) (r = 0.96, P < 0.0008). When infected tissue samples were analyzed by cell culture and PCR, the correlation coefficient between IFUs and chlamydial genomes was higher with C. psittaci B577 FRET PCR (r = 0.90, P < 0.0004) than with Chlamydia omp1 SYBR Green PCR (r = 0.85, P < 0.002).

Recombinant bovine adenovirus type 3 expressing bovine viral diarrhea virus glycoprotein E2 induces an immune response in cotton rats.

Recombinant bovine adenovirus is being developed as a live vector for animal vaccination and for human gene therapy. In this study, two replication-competent bovine adenovirus 3 (BAV-3) recombinants (BAV331 and BAV338) expressing bovine viral diarrhea virus (BVDV) glycoprotein E2 in the early region 3 (E3) of BAV-3 were constructed. Recombinant BAV331 contains chemically synthesized E2 gene (nucleotides modified to remove internal cryptic splice sites) under the control of BAV-3 E3/major late promoter (MLP), while recombinant BAV338 contains original E2 gene under the control of human cytomegalovirus immediate early promoter. Since E2, a class I membrane glycoprotein, does not contain its own signal peptide sequence at the 5' end, the bovine herpesvirus 1 (BHV-1) glycoprotein D signal sequence was fused in frame to the E2 open reading frame (ORF) for proper processing of the E2 glycoprotein in both the recombinant viruses. Recombinant E2 protein expressed by BAV331 and BAV338 recombinant viruses was recognized by E2-specific monoclonal antibodies as a 53-kDa protein, which also formed dimer with an apparent molecular weight of 94 kDa. Insertion of an E2-expression cassette in the E3 region did not effect the replication of recombinant BAV-3s. Intranasal immunization of cotton rats with these recombinant viruses generated E2-specific IgA and IgG responses at the mucosal surfaces and in the serum. In summary, these results show that the pestivirus glycoprotein can be expressed efficiently by BAV-3. In addition, mucosal immunization with replication-competent recombinant bovine adenovirus 3 can induce a specific immune response against the expressed antigen.

Evidence for a parapox ovis virus-associated superantigen.

As shown in a number of species, susceptibility to infectious diseases can be efficiently reduced following application of inactivated parapox ovis viruses (iPPOV). However, the basic mechanism for this stimulating capacity of iPPOV remains unclear. When analyzed, the interaction of iPPOV with porcine peripheral blood mononuclear cells was seen to involve T helper cells as the main target cell population responding to iPPOV. These cells displayed a strong proliferation, and were the major source for the observed increased levels of IL-2. Activation of the T helper cells was MHC class II dependent, but not MHC class II restricted: cellular processing of iPPOV was not required for presentation by autologous, allogeneic or xenogeneic MHC class II molecules. Furthermore, CD3 and CD4 molecules were involved in the stimulation, indicating a receptor-mediated activation of T helper cells. The results demonstrated typical characteristics of a superantigen-induced response providing evidence for a viral component within PPOV functioning as superantigen(s) in swine.

Poxvirus-induced immunostimulating effects on porcine leukocytes.

The prophylactic application of inactivated parapox ovis viruses (Baypamun; Bayer AG, Leverkusen, Germany) has been shown to reduce efficiently the outbreak of stress-mediated diseases in different species. However, little is known about the basic mechanism behind this observed stimulatory property. We therefore tested eight inactivated poxvirus strains belonging to three different genera (Orthopoxvirus, Avipoxvirus, and Parapoxvirus) for their capacity to activate cells of the porcine innate and specific immune systems in vitro. The results indicated that poxviruses failed to induce increased phagocytosis, oxidative burst, or natural killer cell activity in swine. In contrast, enhanced release of interleukin-2, alpha interferon, and gamma interferon, as well as strong proliferation, could be measured. Flow cytometric analyses and cell sorting experiments identified T-helper cells as the main target responding to inactivated poxviruses: the activated cells had a CD4(high) CD25(+) major histocompatibility complex type II-positive phenotype and were the major source of secreted cytokines. Together, the results demonstrated that all tested poxviruses possessed immunostimulating capacity. These in vitro poxvirus-induced effects may be responsible at least in part for the in vivo immunostimulating capacity of inactivated poxviruses.

Shiga toxin 1 from Escherichia coli blocks activation and proliferation of bovine lymphocyte subpopulations in vitro.

Shiga toxin-producing Escherichia coli (STEC) is widespread in the cattle population, but the clinical significance of Shiga toxins (Stx's) for the bovine species remains obscure. Since Stx's exert immunomodulating effects in other species, we examined the effect of purified Stx1 on a bovine B lymphoma cell line (BL-3) and peripheral blood mononuclear cells (PBMC) isolated from adult bovine blood by viability assays and flow cytometry analysis. Stx1 markedly induced apoptosis in stimulated BL-3 cells. The susceptibility of this B-cell-derived cell line was induced only by either lipopolysaccharide (LPS) or pokeweed mitogen, while cultures stimulated with T-cell mitogens were unaffected by the toxin. In contrast, Stx1 did not induce cellular death-neither apoptosis nor necrosis-in primary cultures of PBMC but hindered the mitogen-induced increase in metabolic activity. The influence of Stx1 on single PBMC subpopulations varied with the type of mitogenic stimulus applied. Stimulation with phytohemagglutinin P particularly induced the proliferation of bovine CD8-expressing (BoCD8(+)) cells, and this proliferative response was blocked by Stx1. On the other hand, Stx1 reduced the portion of viable B cells in the presence of LPS. Modulation of activation marker expression (BoCD25 and BoCD71) by Stx1 indicated that the toxin hindered the proliferation of cells by blocking their activation. In conclusion, we assume that Stx1 contributes to the pathogenesis of STEC-associated diarrhea in calves by suppressing the mucosa-associated immune response. The usefulness of cattle as a model in which to study Stx-induced immunomodulation is discussed.

Controlled multiplex PCR of enterotoxigenic Clostridium perfringens strains in food samples.

A controlled multiplex polymerase chain reaction (PCR) for the detection of Clostridium(C.) perfringens enterotoxin gene (cpe) was established and compared with an in vitro antigenic detection method. Thecpe PCR and the classical method of electric immunodiffusion gave identical results. The predicted specific amplicon of the cpe gene was generated from all of the tested enterotoxigenic C. perfringens strains. In contrast, cultures of any other Clostridium species tested by PCR were negative (100% sensitivity, 100% specificity). Addition of an alphatoxin (plc) gene specific PCR as an in process control reaction was performed in order to prevent false negative PCR results. The PCR detection limit was 0.5 ng of genomic C. perfringens DNA per ml of bouillon culture. By contaminating minced meat with C. perfringens reference strains, the multiplex PCR was established as a tool for routine diagnostic laboratories. The detection limit was approximately 3.0x10(5)C. perfringens cells per gram meat. The results demonstrate the multiplex PCR as an easy, specific, sensitive and time saving diagnostic procedure. Application of this improved method should enhance the knowledge concerning epidemiological aspects of food borne diseases caused by enterotoxigenic C. perfringens.

Neutralization of hemolytic and mouse lethal activities of C. perfringens alpha-toxin need simultaneous blockade of two epitopes by monoclonal antibodies.

Three murine monoclonal antibodies (MAbs 3B4, 1E8, 1F9) were produced by fusion of X63-Ag8.653 myeloma cells and splenocytes of mice immunized with glutaraldehyde-inactivated alpha-toxin of Clostridium perfringens. All MAbs belonged to the immunoglobulin G (IgG) class and possessed a kappa light chain. All the MAbs were specific for alpha-toxin of C. perfringens as demonstrated by immunoblotting experiments performed with culture supernatants of C. perfringens, C. bifermentans, C. sordellii, and Bacillus cereus. Competition analysis in an ELISA revealed that the MAbs recognized different epitopes on the alpha-toxin molecule. In an immunoblot assay based on a recombinant protein expressed in Escherichia coli, the binding site of MAb 1E8 but not those of MAbs 3B4 and 1 F9 were mapped to the COOH-terminal fragment of alpha-toxin (aa 248-370). To prove the neutralizing potential of the MAbs, alpha-toxin was preincubated with MAbs and subsequently tested for its lecithinase activity in an egg yolk diffusion turbidity (EYDT) assay, its hemolytic activity in a hemolysis test, and its lethal effect on mice after intraperitoneally administration. When the MAbs were tested individually, neutralization was only seen in the EYDT assay, where MAb 3B4 completely abolished the lecithinase activity of alpha toxin. However, when MAbs 3B4 and 1 E8 were used in combination, they acted synergistically and inhibited the lysis of rabbit erythrocytes in vitro. The same mixture of MAbs was also able to completely neutralize the lethal effect of three LD50 of alpha-toxin on Balb/c mice. Our results suggest that the alpha-toxin molecule contains several domains which are differently involved in the various activities of the toxin. We conclude that the hemolytic domain(s) of alpha-toxin is (are) identical with or very closely located to the domain(s) that cause the mouse lethal effect. The lecithinase activity may be involved in the mechanisms of hemolysis and mouse lethality but appears not to be the only determinant.

The use of Baypamun N in crowding associated infectious respiratory disease: efficacy of Baypamun N (freeze dried product) in 4-10 month old horses.

The efficacy of an immunomodulator, Baypamun N, was tested in 4-10-month-old horses which were exposed to stress by weaning, transport and commingling with yearlings from different breeders (crowding). Verum (n = 26) and placebo animals (n = 27) received three intramuscular injections of the investigational preparations (days 0, 2, 9) starting at the day of commingling in one stable. The incidence of acute respiratory disease was high during the first 4 weeks after commingling. Approximately 50% of all horses showed seroconversion due to field infection by EHV1 and EHV4 during the observation period. The clinical scores in the Baypamun N group were significantly reduced by 40.3% (P < 0.05) compared to the placebo group. The proportion of horses with purulent nasal discharge during the observation period (4 weeks) was also significantly reduced by 58.7% (P < 0.01) in the Baypamun N group. Fifty per cent of the horses injected with Baypamun N showed no purulent nasal discharge and therefore no signs of complicated disease of the upper respiratory airways in contrast to only 14.8% in the non-protected placebo group. The challenge conditions studied in this investigation were rather severe because of the permanent exposure of Baypamun N treated individuals to the non-separated and untreated horses (n = 51). This indicates that treatment with Baypamun N is a successful tool to avoid severe clinical consequences of stress in young horses.

Shiga toxin-producing Escherichia coli strains from bovines: association of adhesion with carriage of eae and other genes.

Out of 174 bovine Shiga toxin-producing Escherichia coli (STEC) strains isolated from diarrheic calves in Germany and Belgium, 122 strains (70.1%) were selected because of their reactivity with the eae (E. coli attaching and effacing gene) probe ECW1-ECW2. One hundred seven of these eae-positive strains (87.7%) harbored stx1 genes, 13 strains (10.7%) had stx2 genes, and 2 strains (1.6%) had both stx genes. The strains displayed 17 different O types, the majority (97 strains) [79.5%]) belonging to O5 (5 strains), O26 (21 strains), O111 (13 strains) O118 (36 strains), O145 (9 strains), and O157 (13 strains). In the HEp-2 cell adhesion assay, 99 strains (81.1%) showed a localized adhesion, and 80 strains (65.6%) stimulated actin accumulation, as determined in the fluorescence actin staining test. None of the strains harbored genes coding for bundle-forming pili (bfpA), clearly differentiating them from enteropathogenic. E. cole. espB gene sequences were only detectable in 23 (18.9%) of the eae-positive bovine STEC strains. Three different PCRs were established, differentiating between eae sequences of enteropathogenic E. coli strain E2348/69 (O127:H6) and STEC strain EDL933 (O157: H7). Primers matching in the more heterologous downstream eae sequences gave amplicons in only 8 of the 17 O types (O84:H-, O103:H2, O111:H-, O111:H2, O119:H25, O128:H-, O145:H28, and O157:H-). Only 15 STEC strains, belonging to serotypes O111H:-, O111H:2, O145:H28, and O157:H-, gave amplicons in all three eae-specific PCRs. These data demonstrate that bovine STEC strains are a heterogeneous group of pathogenic bacteria, a lot of which share virulence markers with STEC strains causing infections in humans. However, in contrast to human STEC strains, bovine eae-positive STEC strains are mainly restricted to the stx1 genotype. The observation that espB sequences are not highly conserved might have consequences for the serological recognition of the ESPB protein in patients. Like in human STEC strains, eae-related sequences are closely associated with certain E. coli O groups; however, they are not serotype specific.

The enterohemolysin phenotype of bovine Shiga-like toxin-producing Escherichia coli (SLTEC) is encoded by the EHEC-hemolysin gene.

Naturally occurring enterohemolysin negative variants were observed during studies on bovine Shiga-like toxin-producing E. coli (SLTEC). Examination of three strains (413/89-1 and 332, 026:H-, and 570/89, O111:H-) and their isogenic variants (413/89-6, 332-I and 570/89-I, respectively) showed, that in each strain loss of the enterohemolytic phenotype correlated with the loss of a large plasmid ranging from 94 to 104 kb in size. The hemolysin determinant present on the 94 kb plasmid of strain 413/89-1 was cloned and discovered by DNA and N-terminal aminoacid sequence analysis to be highly homologous to the recently published EHEC-hemolysin (HlyEHEC; Schmidt et al., 1994; 1995). When a recombinant plasmid harboring this determinant was reintroduced into the enterohemolysin negative isogenic mutant 413/89-6, the enterohemolytic phenotype was restored. Southern blot hybridization analysis was used to demonstrate that the HlyEHEC is plasmid-borne in SLTEC-strains. Our cumulative data suggest that the enterohemolytic phenotype of SLTEC is encoded by the plasmid-borne HlyEHEC. These results further demonstrate the close similarity between SLTEC-isolates from bovine and human.

The region between the M and S genes of porcine haemagglutinating encephalomyelitis virus is highly similar to human coronavirus OC43.

The nucleotide sequences of the regions between the membrane and spike protein genes of three strains of porcine haemagglutinating encephalomyelitis virus (HEV) were determined. A total of 739 (HEV strain 67N) and 751 (strains NT9 and VW572) nucleotides were sequenced. Two ORFs, potentially encoding proteins of 12.8 and 9.6 kDa, were identified. Pairwise comparisons with the corresponding ORFs in bovine coronavirus (BCV) and human coronavirus (HCV) OC43 revealed sequence similarities of greater than 88.5 percent at the nucleotide and 85.3 percent at the amino acid level for the 12.8 kDa ORF product. For the 9.6 kDa ORF product similarities were greater than 96.9 percent and 95.2 percent, respectively. An additional 12 nucleotide deletion upstream of the 12.8 kDa ORF start codon was found in HEV 67N compared to NT9 and VW572. These results reveal a genomic organization of HEV in the region analysed that is homologous to HCV OC43 but different from BCV.

Synthesis and evaluation of a non-radioactive gene probe for the detection of C.perfringens alpha toxin.

The synthesis and evaluation of a non-radioactive hybridization probe is described, specific detecting the Clostridium perfringens alphatoxin gene (plc) by colony blot hybridization assay. A vector free digoxigenin-dUTP-labelled probe was generated by polymerase chain reaction (PCR) targeting the cloned plc gene of C.perfringens strain ATCC 13124. In a colony blot hybridization assay 296 strains of C.perfringens were tested for plc. None of the strains failed in hybridization. Presence of plc was even demonstrated in C.perfringens strains reported to lack lecithinase activity. Specificity of the probe was shown with various strains of other bacterial species. None different Clostridia sp. tested, e.g. C.bifermentans, C.tertium, C.novyi, C.chauvoei, C.sporogenes, C.difficile, C.putrifucum, C.sordellii, C.botulinum, C. septicum and C.histolyticum, hybridized with the plc specific probe. Strains expressing an enzymatically related phospholipase like Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus gave also negative results. Comparing the results of conventionally used egg yolk turbidity assay and those gained with DNA hybridization, the plc probe proved to be a much more sensitive and specific diagnostic tool for the detection of C.perfringens plc.

Genomic relationship of porcine hemagglutinating encephalomyelitis virus to bovine coronavirus and human coronavirus OC43 as studied by the use of bovine coronavirus S gene-specific probes.

The genomic relationship of porcine hemagglutinating encephalomyelitis virus (HEV) to bovine coronavirus (BCV) and human coronavirus (HCV) strain OC43 was examined by dot blot hybridization assays. Two BCV S gene-specific probes were generated by polymerase chain reaction from the avirulent L9-strain of BCV. Probes were located in the S1 and the S2 region of the peplomeric (S) glycoprotein gene. The S1 probe (726 bp) hybridized with BCV and HCV-OC43, but not with HEV under moderate stringency hybridization conditions (50 degrees C). Only slight signals were present with mouse hepatitis virus (MHV) and no signals were observed with feline infectious peritonitis virus (FIPV) or canine coronavirus (CCV). At high stringency conditions (60 degrees C) the S1 probe hybridized with BCV only. Using the S2 probe (680 bp) under moderate stringency conditions, hybridization signals were obtained with BCV, HCV-OC43 and HEV (strains 67N, NT9, VW572). The signals obtained by the three HEV strains were altogether weaker than with BCV and HCV-OC43. The S2 probe did not react with MHV, FIPV and CCV. At high stringency the S2-specific probe hybridized with BCV and HCV-OC43 but did not hybridize with HEV. Nucleotide sequence analysis of the region covering the S2 probe in HEV revealed 92.6% nucleotide sequence homology to BCV and 91.9% to HCV-OC43. In contrast, the region covering the S1 probe in HEV could not be amplified using the BCV S1-specific primers. The hybridization and sequencing results thus indicate a closer genomic relationship between BCV and HCV-OC43 than there is between HEV and BCV or HCV-OC43 respectively.

Transcription of two divergently transcribed yeast genes initiates at a common oligo(dA-dT) tract.

Oligo(dA-dT) tracts are frequently found in the intergenic regions of the yeast Saccharomyces cerevisiae and have been proposed to act as upstream promoter elements for constitutive transcription. An oligo(dA-dT) tract of 23 bp is also found as a characteristic sequence motif in the centre of the 230 bp segment which separates the open reading frames of the CBS2 gene and its 5'-flanking gene on chromosome IV. Recently we have reported that transcription of CBS2 is initiated immediately adjacent to this oligo(dA-dT) tract (michaelis et al. 1988). Here we report that the flanking gene of unknown function is divergently transcribed into an RNA with heterogeneous 5' ends. Two of these 5' ends map within the oligo(dA-dT) stretch, while the third is located upstream, leading to an RNA species which is partially complementary to the CBS2 transcript. Gel shift assays show that the oligo(dA-dT) stretch is specifically recognized by (a) binding factor(s) in nuclear extracts. We discuss these results with respect to the role of oligo(dA-dT) stretches in gene expression in yeast.

Yeast nuclear gene CBS2, required for translational activation of cytochrome b, encodes a basic protein of 45 kDa.

In yeast, synthesis of apocytochrome b from mitochondrial COB mRNA depends on at least three nuclear gene products. The translation stimulatory effect by two of these nuclear genes, CBS1 and CBS2, is mediated by the 5'-untranslated leader of COB mRNA. In this report, we show that CBS2 is located on chromosome IV and provide genetic evidence that the CBS2 gene encodes a polypeptide. Determination of the DNA sequence reveals a contiguous open reading frame of 1167 bp. The deduced polypeptide has a calculated molecular weight of 44.5 kDa and is characterized by a high content of positively charged amino acids. It has no significant homology to any known protein. The CBS2 gene is transcribed into low abundance mRNA species with a major transcription initiation site located 97 bp upstream from the ATG start codon next to a poly(dA-dT) stretch.