Replicon particle vaccine protects swine against influenza.

An alphavirus derived replicon particle (RP) vaccine expressing the cluster IV H3N2 swine influenza virus (SIV) hemagglutinin (HA) gene induced protective immunity against homologous influenza virus challenge. However, pigs with maternal antibody had no protective immunity against challenge after vaccination with RP vaccines expressing HA gene alone or in combination with nucleoprotein gene.

Identification of a putative cellular receptor for bovine herpesvirus 1.

Recognition of a host cell receptor by a virus is the first and perhaps the most crucial step in initiating the disease process. This study was undertaken to identify the cellular receptor(s) for bovine herpesvirus 1 (BHV-1). Previously, we reported the development and characterization of bovine anti-idiotypic antibodies (anti-ids) that induce neutralizing antibodies to BHV-1. These anti-ids inhibit BHV-1 penetration of permissive cells. We have used these anti-ids, which mimic an epitope on the virus glycoprotein IV (gIV), and gradient-purified virus in immunoprecipitation (IP) as well as photoaffinity labelling (PAL) assays. In the IP assays, both bovine anti-ids and BHV-1 virions coupled to Sepharose precipitated a 60K protein from 125I-labelled BHV-1 permissive cell membrane extracts. Normal bovine IgG or an irrelevant virus, transmissible gastroenteritis virus (TGEV), used as negative controls failed to precipitate this protein. Similarly, in the PAL assays, the 60K cell surface protein was identified on cells permissive for BHV-1 infection, but not on non-permissive cells when 125I-labelled ligands, the anti-ids or BHV-1 were used as probes. The iodinated ligands failed to identify the 60K protein if they had been pretreated with the antibody 1. Pretreatment of the iodinated ligands with an isotype-matched control antibody had no effect on the identification of the 60K protein present on cells permissive for BHV-1 infection. The negative controls, i.e. normal bovine IgG and TGEV, failed to identify this 60K protein on permissive or non-permissive cells. These results suggest that the 60K protein is a cellular receptor recognized by BHV-1 during the infection process.

Comparison of serum responses in swine after vaccination and challenge exposure with Actinobacillus pleuropneumoniae serotype 1.

Clinical trials have shown that currently available commercial vaccines against porcine pleuropneumonia provide inconsistent, serotype-specific protection from the disease. Recovery from naturally acquired infection, however, provides solid, serotype cross-protective immunity. We examined various serum responses of pigs receiving 1 of 4 commercial vaccines or a cell extract, and compared the serologic responses of these pigs after challenge exposure with virulent Actinobacillus pleuropneumoniae serotype 1. Evaluation of serum included complement-mediated killing, opsonizing capacity, IgG titers to whole organisms, and cytotoxin neutralization titers. Pigs that received the cell extract had fewer clinical signs of pleuropneumonia than pigs in other vaccinated groups, and also were significantly (P < 0.05) better protected from development of lung lesions and death. Such vaccinates were the only pigs that developed significant (P < 0.05) serum antibody titers (ie, protective immune response) to whole-cell antigens and to cytotoxin.

The importance of secreted virulence factors in Actinobacillus pleuropneumoniae bacterin preparation: a comparison.

Current bacterins provide only partial protection against morbidity and mortality in swine following infection by Actinobacillus pleuropneumoniae. We compared the efficacy of a cell-free concentrate from mid-log phase growth cultures of Actinobacillus pleuropneumoniae (APP) serotype 1 to four commercial bacterins. This cell-free preparation contained carbohydrate, endotoxin, and protein, and had hemolytic and cytotoxic activity. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis analysis indicated the presence of one major 110,000-molecular-weight protein. This protein band also stained by the periodic acid Schiff method, indicating the presence of carbohydrate. Cell-free concentrates of APP serotypes 5 and 7 had identical profiles following electrophoresis and staining with either Coomassie blue for protein or Schiff reagent for carbohydrate. Lipopolysaccharide profiles for the cell-free concentrates of serotypes 1 and 5 were semi-rough while the LPS profile for serotype 7 was smooth. Five A. pleuropneumoniae-free SPF pigs per group were vaccinated on days 0 and 21 with cell-free concentrate of serotype 1 plus adjuvant, or one of four commercial bacterins according to the manufacturer's directions. Control pigs were vaccinated with PBS mixed with adjuvant. All pigs were challenged intranasally on day 35 with serotype 1 and necropsied on day 50. Protection was greatest in the cell-free concentrate group, as compared with all other groups, in that no deaths occurred, clinical scores were less severe, and percent lung affected was significantly reduced (P < 0.05). In addition, whole-cell ELISA titers were significantly increased (P < 0.05) postvaccination in the cell-free concentrate group, and postvaccination and postchallenge sera neutralized the hemolytic activity of the cell-free concentrate from serotypes 1 and 5 (P < 0.05), as compared with all other groups. No serum neutralization to the hemolysin of serotype 7 was observed. Immunoblot analysis using antisera derived from gnotobiotic pigs indicated that the cell-free vaccine generated a response that was identical to the response observed following live challenge. Similar, but not identical, responses were observed when antisera generated against the bacterins was used. This study indicates that an acellular vaccine containing multiple virulence factors can provide complete protection from mortality and significantly reduced morbidity to homologous challenge.

Actinobacillus pleuropneumoniae-induced thymic lesions in mice and pigs.

Actinobacillus pleuropneumoniae produces several hemolysins/cytotoxins that may be important in the pathogenesis of acute lesions. Little is known, however, about the role of these virulence factors in chronic disease or the carrier state. We investigated the effects of live bacterial infection and transthoracic injection of a sterile culture supernatant on primary lymphoid organs and lymphocyte populations. Transthoracic inoculation of mice or intranasal inoculation of pigs with virulent A. pleuropneumoniae serotypes 1 and 7 induced thymic cortical lymphoid necrosis. These lesions were reproduced in mice by transthoracic injection of a concentrated sterile culture supernatant. The cytotoxic effect of this culture supernatant was also demonstrated in vitro by using a tetrazolium dye reduction assay. Both porcine and murine thymic lymphocytes as well as splenic T lymphocytes were susceptible to the toxin. Porcine convalescent serum, but not preimmune serum, prevented thymic lesions and neutralized the in vitro cytotoxic effect of the culture supernatant on murine thymic lymphocytes. Thymic lesions also were reproduced in mice by using purified lipopolysaccharide (LPS) from Escherichia coli O111:B4; however, LPS had no in vitro cytotoxic effect on either porcine or murine thymic lymphocytes. These results suggest that secreted A. pleuropneumoniae toxin(s) is capable of affecting host T-lymphocyte populations and may affect host immune function.

Efficacy of a cell extract from Actinobacillus (Haemophilus) pleuropneumoniae serotype 1 against disease in swine.

We partially characterized a cell extract (CE) from Actinobacillus pleuropneumoniae serotype 1 and used the CE to test the efficacy of secreted proteins against disease. Secreted products from 4-h culture supernatants were precipitated with 20% polyethylene glycol. Analysis of the CE indicated the presence of protein, endotoxin, and carbohydrate. Hemolytic activity to bovine erythrocytes and cytotoxic activity to porcine mononuclear leukocytes was also demonstrated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the CE from a 4-h culture showed a major band at 110 kilodaltons (kDa), while a CE of a 26-h culture indicated the presence of a number of additional proteins, including the 110-kDa protein. The 110-kDa protein was also identified as a glycoprotein by periodic acid-Schiff and silver staining. A single band precipitated against convalescent-phase pig antiserum when the polyethylene glycol precipitate was used in an Ouchterlony plate. Vaccination with CE conferred greater protection against challenge with the homologous serotype than either a commercial bacterin or an outer membrane protein vaccine. Hemolysin-neutralizing titers were higher both pre- and postchallenge in the group vaccinated with the CE compared with in all other groups. We believe that this demonstrates the importance of secreted factors in protection against disease and suggests that the 110-kDa protein is an important immunogen.