Clostridium (C.) difficile is a common cause of nosocomial diarrhea in horses. Vancomycin and metronidazole have been used as standard treatments but are only moderately effective, which highlights the need for a novel alternative therapy. In the current study, we prepared antiserum of equine origin against both C. difficile toxins A and B as well as whole-cell bacteria. The toxin-neutralizing activities of the antibodies were evaluated in vitro and the prophylactic effects of in vivo passive immunotherapy were demonstrated using a conventional mouse model. The data demonstrated that immunized horses generated antibodies against both toxins A and B that possessed toxin-neutralizing activity. Additionally, mice treated with the antiserum lost less weight without any sign of illness and regained weight back to a normal range more rapidly compared to the control group when challenged orally with 10(7) C. difficile spores 1 day after serum injection. These results indicate that intravenous delivery of hyperimmune serum can protect animals from C. difficile challenge in a dose-dependent manner. Hence, immunotherapy may be a promising prophylactic strategy for preventing C. difficile infection in horses.
Animals ; Antibodies, Bacterial/blood/*immunology/therapeutic use ; Bacterial Proteins/immunology/therapeutic use ; Bacterial Toxins/immunology/therapeutic use ; Clostridium Infections/microbiology/prevention & control/*veterinary ; Clostridium difficile/*immunology ; Enterotoxins/immunology/therapeutic use ; Female ; Horse Diseases/microbiology/*prevention & control ; Horses ; Immune Sera/*immunology ; Immunization, Passive/*veterinary ; Mice ; Mice, Inbred C57BL ; Spores, Bacterial/immunology

OBJECTIVETo synthesize two antigens-Ag85b and HspX of Mycobacterium tuberculosis H37Rv with molecular biological methods and to observe their biologic activity after co-administration of adjuvants (aluminum and/or CpG) in mice.

METHODSRecombinant expression plasmids pET30a-Ag85b and pET30a-HspX were constructed. The objective DNA fragments was characterized with restriction enzyme. Then the recombinant plasmids were transformed into E. coli BL-21, and two proteins were expressed by induction of isopropyl beta-D-1-thiogalactopyranoside. After purification with anion exchange column Source30, QHP, and hydrophobic chromatography column, two proteins were identified by amino acid sequencing. After the successful preparation of these two antigens, they were co-administered in mice with adjuvants of aluminum and/or CpG (Ag85b, Ag85b + Al, Ag85b + CpG, Ag85b + Al + CpG; HspX, HspX + Al, HspX + CpG, HspX + Al + CpG); one group received normal saline and served as the control. Splenic lymphocytes were isolated for enzyme-linked immunosorbent spot assay to detect the secreted specific interferon-gamma (IFN-gamma); in addition, lymphocytes proliferation test was performed to observe lymphocytes proliferation after in vitro stimulated with two antigens.

RESULTSThe purity of two proteins reached 95% after purification. The N-terminal amino acid sequence (15 aa) of the purified proteins was same as the target sequence. For Ag85b, the secreted specific IFN-gamma from isolated splenic lymphocytes after having been stimulated in vitro with Ag85b (80 microg/ml) remarkably increased in Ag85b + CpG group, Ag85b + Al group, and Ag85b + CpG + Al group; the changes were significantly different between these three groups and control group (P < 0.05). For HspX, the changes were significantly different between HspX + Al + CpG group and normal sodium group, although remarked increase of IFN-gamma was also observed in HspX group, HspX + Al group, and HspX + CpG group.

CONCLUSIONSAg85b and HspX were successfully expressed and purified. A cell-mediated immunity may be induced when the antigens are co-administered with adjuvants of aluminum and/or CpG in mice, indicating that the recombinant proteins are bioactive.

Acyltransferases ; administration & dosage ; isolation & purification ; therapeutic use ; Adjuvants, Immunologic ; therapeutic use ; Animals ; Antigens, Bacterial ; administration & dosage ; isolation & purification ; therapeutic use ; Bacterial Proteins ; administration & dosage ; isolation & purification ; therapeutic use ; Escherichia coli ; Immunity, Cellular ; Interferon-gamma ; Mice ; Mycobacterium tuberculosis ; immunology ; metabolism ; Recombinant Proteins ; administration & dosage ; isolation & purification ; therapeutic use

OBJECTIVESTo assess the efficacy of plasmid DNA encoding pac gene of Streptococcus mutans (S. mutans) intranasally immunized in gnotobiotic rats and to compare the effect of two different delivery systems.

METHODSSprague Dawley rats, infected with S. mutans at 20 days of age, were intranasally immunized with plasmid pCIA-P (group A), Dosper-DNA complex (group B), Bupivacaine-DNA complex (group C). Control rats were either immunized with plasmid pCI (group D), distilled water (group E) or immunized intramuscularly (group F). All the rats were boosted 2 weeks later. ELISA determined the antibodies against the vaccines. Keyes caries score was used to evaluate the anti- caries effectiveness of the vaccines at the terminal study.

RESULTSAs for the antibody reactions, there were significantly (P < 0.01) differences between rats immunized with DNA vaccine and non-immunized rats. And rats in group B and C had the significantly (P < 0.01) higher level of specific salivary anti-PAc IgA antibodies and rats (group B, C, F) had the significantly (P < 0.01) higher specific serum anti-PAc IgG responses to DNA vaccine. Keyes scores of rats (group B and C) were significantly (P < 0.01) lower than others.

CONCLUSIONSIntranasal immunization with plasmid pCIA-P encoding pac gene successfully reduces the caries and appears to be a promising approach against dental caries. Cationic liposome Dosper and local anesthetic bupivacaine could enhance the efficacy of DNA vaccine.

Administration, Intranasal ; Animals ; Antibodies, Bacterial ; blood ; Bacterial Proteins ; genetics ; Dental Caries ; prevention & control ; Female ; Immunization ; Immunoglobulin A ; blood ; Immunoglobulin G ; blood ; Membrane Glycoproteins ; Plasmids ; genetics ; Rats ; Rats, Sprague-Dawley ; Streptococcal Vaccines ; genetics ; immunology ; therapeutic use ; Streptococcus mutans ; genetics ; immunology ; Treatment Outcome ; Vaccines, DNA ; genetics ; immunology ; therapeutic use

OBJECTIVETo construct a fusion anti-caries DNA vaccine pGLUA-P carrying GLU fragment from gtfB gene of Streptococcus mutans GS-5 and A-P fragment including the A region and P region of PAc protein from a DNA anti-caries vaccine pCIA-P, and to investigate its expression in prokaryotic and eukaryotic cells.

METHODSThe sequence of GLU fragment in pGLU plasmid was testified by DNA sequencing. The fusion anti-caries DNA vaccine was constructed by ligating A-P fragment from pCIA-P to pGLU. The expression of GLUA-P fusion protein in E. coli BL21 (DE3) was induced by IPTG and checked by SDS-PAGE electrophoresis. pGLUA-P was transfected in vitro to cultured rat primary muscle cells by cation liposome Dosper, and immunohistochemical method was used to test the expression of GLUA-P fusion protein in cells.

RESULTSGLU sequence was identical with relative sequence of GTF-I (GS-5 strain) in Gene Bank. Recombinant eukaryotic expression plasmid pGLUA-P was confirmed to have both GLU and A-P fragment. After pGLUA-P was transferred into E. coli (DE3), it could express a new 115 000 protein by the induce of IPTG. Specific brown products could be found in the cytoplasm of cultured rat primary muscle cells transfected by pGLUA-P.

CONCLUSIONSFusion anti-caries DNA vaccine pGLUA-P is successfully constructed and confirmed by sequencing and enzymes digestion. Fusion GLUA-P protein can be correctly expressed in prokaryotic and eukaryotic cells.

Animals ; Animals, Newborn ; Bacterial Proteins ; genetics ; metabolism ; Cells, Cultured ; Cloning, Molecular ; Dental Caries ; prevention & control ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; Gene Expression ; Glucosyltransferases ; genetics ; metabolism ; Membrane Glycoproteins ; Muscle, Skeletal ; cytology ; metabolism ; Plasmids ; genetics ; Rats ; Rats, Wistar ; Recombinant Fusion Proteins ; genetics ; metabolism ; Streptococcal Vaccines ; genetics ; immunology ; therapeutic use ; Streptococcus mutans ; genetics ; immunology ; Transfection ; Vaccines, DNA ; genetics ; therapeutic use