Insertion vectors for construction of recombinant conjugative transposons in Bacillus subtilis and Enterococcus faecalis.

The broad-host range of conjugal transfer and the chromosomal location make conjugative transposons (CT) attractive candidates as tools for genetic manipulation of a large variety of bacteria. In this paper we describe insertion vectors capable of integrating into Tn916, the prototype of CT in Gram-positive bacteria. The integration of vectors into a single chromosomal copy of Tn916 was studied both after natural transformation of Bacillus subtilis, and after electroporation in Enterococcus faecalis. Integration occurred either by double or by single crossover, and the integrated DNA segment was shown to be highly stable. All recombinant CT (rCT) were still able to excise from the chromosome to form circular intermediates, the first step of both transposition and conjugal transfer. All classes of rCT generated by insertion vector pSMB47 were capable of conjugal transfer, while using pVMB11 it was possible to generate non-conjugative rCT.

Phenotypic features of BHK-21 cells used for production of foot-and-mouth disease vaccine.

BHK-21 c13 monolayer and suspension cells were investigated with regard to some phenotypic features which could bear on the quality of foot-and-mouth disease virus (FMDV) antigen produced in them. Despite good viability, suspension cells differed from monolayer cells in fundamental features of susceptibility to FMDV. Most important, FMD virus particles grown in suspension cells at high passage levels were shown to be largely degraded following inactivation with an aziridine compound. Suspension cells were characterised by a downregulation in the surface expression of both alpha 5 and alpha V integrin chains. According to the results of binding assays, both integrins could act as FMDV receptors on BHK-21 c13 cells. Reduced surface expression of integrins was correlated with disappearance of actin stress fibres, which would play a role in regular encapsidation of viral RNA and hence in stability of virus particles. With regard to FMD vaccine production, practical suggestions are put forward to evaluate the quality of BHK-21 c13 cells and FMDV Ag, which must prove stable during downstream processing.

Characterization of a distinct subpopulation of bovine gamma delta T cells.

A population of mononuclear cytotoxic cells from peripheral blood leucocytes of cattle showed no usual markers of B and T lymphocytes. However, it could be allocated to a previously unreported gamma delta T cell compartment. This assumption was suggested by: 1. The surface expression of CD3; 2. PCR amplification of the C delta TcR gene from cDNA; and 3. The detection of peripheral blood precursors expressing the workshop cluster (WC) 1 marker of bovine gamma delta T cells. These cells are recognized by murine monoclonal antibodies (mAbs) 5D4, 1E7, 6F9 and 8D7, raised in the authors' laboratory. The above mAbs also identify distinct groups of cells in thymus, spleen, lymph nodes and about 1% of uncultured PBL. The most diffuse infiltration of such cells was shown in the small intestine, as both intraepithelial and lamina propria lymphocytes. Mucosal homing activity was confirmed by immunoperoxidase staining on tongue and pharynx sections of healthy cattle.

Role of a distinct population of bovine gamma delta T cells in the immune response to viral agents.

A distinct population of bovine gamma delta T cells was isolated from peripheral blood mononuclear cells of foot-and-mouth disease (FMD)-vaccinated cattle; these lymphocytes were shown to exert a natural killer-like activity against cells infected by different viruses. The antiviral activity was dependent upon cognate recognition of target cells and could operate by both cytostatic and cytotoxic mechanisms. Among these, secretion of a serine esterase was shown after binding to target cells. This population of bovine gamma delta T cells is recognized by murine monoclonal antibodies 1E7, 5D4, and 6F9, raised in our laboratory. To define an in vivo antiviral role, four heifers were infected with a strain of bovid herpesvirus 1 by the intranasal/intravaginal routes and contact exposure. The prevalence of 1E7+/5D4+ cells among peripheral blood lymphocytes increased dramatically in the first days after infection; the same held true for in-contact cattle, albeit with a different time kinetics. In another experiment, colonization of mucosae was demonstrated by immunoperoxidase staining on tongue and palate sections of healthy cattle. The infiltration of gamma delta T cells altogether in the palate mucosa was much more accentuated in foot-and-mouth disease-vaccinated, as compared to nonvaccinated, control calves.

Electrotransformation of Streptococcus agalactiae with plasmid DNA.

A protocol for efficient electrotransformation of Streptococcus agalactiae (group B streptococcus) Lancefield's strain O90R (NTCT 9993) (an unencapsulated derivative of type Ia strain O90) was developed. The Escherichia coli-Streptococcus shuttle vector pDP28 (7.8 kb) carrying the ermB gene for resistance to erythromycin was used as donor DNA. Frozen 'electrocompetent' cells were prepared by repeated washes in 10% glycerol. A 50-microliters aliquot containing about 5 x 10(9) colony forming units of bacteria was subjected to the electric pulse. Optimal conditions for electrotransformation were determined using different media, harvesting cells at different points of the growth curve, and using different field strengths. The dose-response curve for transformation of S. agalactiae with pDP28 showed one-hit kinetics as donor DNA varied between 0.01 and 3 micrograms. The efficiency of electrotransformation for this range of amounts of donor DNA was 1.2 x 10(4) cfu micrograms-1. The transformation frequencies obtained with this electroporation protocol are high enough to allow both subcloning and shotgun cloning of streptococcal DNA in S. agalactiae.

Immunogenicity of foot-and-mouth disease virus grown in BHK-21 suspension cells. Correlation with cell ploidy alterations and abnormal expression of the alpha 5 beta 1 integrin.

BHK-21 suspension cells were characterized with regard to genetic and phenotypic features which might adversely affect the immunogenic properties of foot-and-mouth disease virus (FMDV) grown therein. A positive correlation was found between number of passages in suspension culture and both prevalence of polyploid cells and reduced cell growth on surfaces. Suspension cells also revealed differences in the expression of RGD-specific integrins and, in particular, of alpha 5 beta 1, which was shown to work as an FMDV receptor structure. These features, along with the notable instability of a few non-structural FMDV A5 proteins in infected cells, outline a new scenario, in which the reduced immunogenicity of FMDV might be accounted for by defined negative influences of the cell environment on viral replication.

Human lymphoblastoid interferon as vaccine adjuvant in cattle.

Human lymphoblastoid interferon from Namalwa cells was purified for clinical use by ethanol fractionation, and used as adjuvant of an inactivated Bovid Herpesvirus 1 vaccine in calves. In agreement with other in vitro and in vivo models, low and high interferon doses were shown to increase and depress the specific antibody response, respectively. The low, effective interferon dose (100 International Units/kg) also reduced the variability of antibody titres after the first vaccine injection. This latter dose had apparently no influence on the regulatory T cell circuits, as opposed to the other doses under study.

Genotypic and phenotypic changes of BHK-21 cells grown in suspension cultures.

The propagation of foot-and-mouth disease virus on BHK-21 suspension cells, although economically convenient, may yield a scarcely immunizing antigen. Helpful insights were obtained by investigating a few genotypic and phenotypic features of the cell cultures. The appearance of polyploid populations, higher cell concentrations at the end of culturing, the progressive reduction of spreading on surfaces and an abnormal expression of the alpha 5 beta 1 integrin were found to be correlated with the number of passages in suspension culture. The observed modifications in the normal course of the cell-cycle and in the expression of some surface proteins point at a possible mechanism of virus damages arising from defective cellular functions.

Genotypic and phenotypic changes of BHK-21 cells grown in suspension cultures.

The propagation of foot-and-mouth disease virus on BHK-21 suspension cells, although economically convenient, may yield a scarcely immunizing antigen. Helpful insights were obtained by investigating a few genotypic and phenotypic features of the cell cultures. The appearance of polyploid populations, higher cell concentrations at the end of culturing, the progressive reduction of spreading on surfaces and an abnormal expression of the α5ß1 integrin were found to be correlated with the number of passages in suspension culture. The observed modifications in the normal course of the cell-cycle and in the expression of some surface proteins point at a possible mechanism of virus damages arising from defective cellular functions.

Target recognition by bovine mononuclear, MHC-unrestricted cytotoxic cells.

A population of bovine non B/non T, cytotoxic lymphocytes with natural killer activity against virus-infected and non-infected embryonic kidney cells was functionally characterized. The data obtained in experiments of flow cytometry and immuno-peroxidase staining show that a CD2-, CD4-, CD8-, TcR gamma delta-, CD3+, CD45+, FcR+ lymphoid killer cell does exist within bovine peripheral blood leucocytes. This population can detect the down-regulation of class I MHC antigens or the expression of embryonic forms thereof, as shown by experiments of 17-hour 51Cr release and binding to target cells. This model was tested in vitro in experiments on virus-infected bovine kidney cells. The emerging picture was substantially in agreement with the "missing self" theory as a possible option for target cell recognition. In this respect, the profound alteration of MHC Class I expression could represent a major early event, recognized on virus-infected cells by the immune system.

Isolation of mononuclear cytotoxic cells from cattle vaccinated against foot-and-mouth disease.

Mononuclear cells from peripheral blood leucocytes of foot-and-mouth disease (FMD) vaccinated cattle underwent blast transformation after in vitro culture with purified, inactivated, 146 S FMD virus antigen. From the prolonged culture of blast cells in medium with Interleukin-2 (5-10 U/ml), CD 45-positive effector cells were derived, which showed a potent, non MHC-restricted activity against virus-infected cells, the extent of which was inversely correlated with multiplicity of infection (MOI). Extensive characterization of effector cells by means of monoclonal antibodies (MAbs) in flow cytometry, lysis of various cell populations with MAbs and complement, Fc receptor analysis and 51Cr release assays indicated that the isolated cells share some phenotypic and functional features of the human and murine Large Granular Lymphocyte/Natural Killer (N.K.) lineage.

Comparative evaluation of specific vaccines and immuno-modulators in disease control of beef cattle.

Two field trials were undertaken to evaluate specific vaccines and Biological Response Modifiers (B.R.M.) with regard to disease control of beef cattle. Clinical data and laboratory results strongly suggested a genetically determined resistance to transportation stress of some cattle breeds, with possible important consequences on the occurrence of multi-factor diseases. Plenty of calves showed detectable levels of serum interferon and a remarkable activation of at least some lymphocyte populations in the first days after arrival. In general, B.R.M.-treated cattle could better react against environmental pathogens, as evidenced by both clinical and laboratory findings. Several data emerged that even the efficacy of specific vaccines could be largely accounted for by a positive modulation of non-specific immunity; this latter feature can be mainly referred to adjuvants and/or some bacterial antigens.