OBJECTIVETo establish a mouse model of humoral immune response by immunization with rabbit red blood cells (RRBCs).

METHODSThe mice were immunized with RRBCs and the serum hemolysin level was measured by micro-hemolysis spectrophotometry.

RESULTSThe peak time needed for hemolysin production against RRBCs was 6 days after the immunization, and 20% RRBCs in a total volume of 0.2 ml was optimal for intraperitoneal injection. Hydrocortisone (25 mg/kg) and cyclophosphamide (20 mg/kg) inhibited hemolysin production. Mannatide (4 mg/kg) produced no significant effect on serum hemolysin level in normal mice, but significantly potentiated hemolysin production in immunosuppressed mice induced by cyclophosphamide (20 mg/kg).

CONCLUSIONIntraperitoneal RRBC injection is feasible for establishing mouse models of humoral immune response.

Animals ; Erythrocytes ; immunology ; Female ; Guinea Pigs ; Hemolysin Proteins ; blood ; Immunity, Humoral ; Immunization ; Male ; Mice ; immunology ; Models, Animal ; Rabbits

Listeria monocytogenes (Lm) is zoonotic pathogen that can cause listeriosis, and vaccine is one of the effective methods to prevent this pathogen infection. In this study, we developed a novel vaccine that is a mixture of inactivated bacteria and Montanide™ ISA 61 VG, a mineral oil adjuvant, and evaluated the safety and immune response characteristics of this vaccine. The mice immunized with the ISA 61 VG adjuvant had high safety, and it could induce significantly higher titer of anti-listeriolysin O (LLO) antibody and higher value of IgG2a/IgG1 ratio compared with the group without the adjuvant. In particular, it could provide 100% immune protection against lethal doses of Lm challenge in mice. In summary, ISA 61VG adjuvant significantly enhanced the ability of inactivated listeria vaccine to induce humoral and cellular immune responses, thereby enhanced the protective immune response in the host, and it is a potential vaccine candidate for the prevention of Lm infection in humans and animals.
Adjuvants, Immunologic ; pharmacology ; Animals ; Hemolysin Proteins ; immunology ; pharmacology ; Immunity, Cellular ; drug effects ; Listeria monocytogenes ; immunology ; Listeriosis ; prevention & control ; Mice ; Mice, Inbred BALB C ; Vaccines, Inactivated ; immunology

Corn, one of the most important forage crops worldwide, has proven to be a useful expression vehicle due to the availability of established transformation procedures for this well-studied plant. The exotoxin Apx, a major virulence factor, is recognized as a common antigen of Actinobacillus (A.) pleuropneumoniae, the causative agent of porcine pleuropneumonia. In this study, a cholera toxin B (CTB)-ApxIIA#5 fusion protein and full-size ApxIIA expressed in corn seed, as a subunit vaccine candidate, were observed to induce Apx-specific immune responses in mice. These results suggest that transgenic corn-derived ApxIIA and CTB-ApxIIA#5 proteins are potential vaccine candidates against A. pleuropneumoniae infection.
Actinobacillus Infections/microbiology/*prevention & control ; Actinobacillus pleuropneumoniae ; Animals ; Antigens, Bacterial/immunology ; Bacterial Proteins/*immunology ; Bacterial Vaccines/*immunology ; Cholera Toxin/*chemistry ; Female ; Hemolysin Proteins/*immunology ; Immunization, Secondary ; Mice ; Mice, Inbred ICR ; Plants, Genetically Modified ; Zea mays/*genetics

OBJECTIVETo investigate the etiological agent of patients with diarrhea followed by acute kidney failure symptoms in China, 1999.

METHODSWestern blot was used to detect serum specific antibodies of patients against entero-haemorrhagic Escherichia coli hemolysin (EHEC-Hly) and lipo-polysaccharide of E. coli O157.

RESULTSTwenty-one and 16 of 42 patients showed positive reaction of specific IgG or IgM antibodies against EHEC-Hly respectively. Eleven of 42 serum samples were positive for having both IgG and IgM antibodies while 26 of 42 samples were positive for IgG or IgM. For E. coli O157 LPS test, 24 and 24 of 42 samples showed positive for IgG or IgM antibodies respectively. In 42 samples, 20 were positive for IgG and IgM while 29 were positive for IgG or IgM.

CONCLUSIONSTwenty-two of 42 samples were reacted with EHEC-Hly and E. coli O157 LPS, but 34 of 42 samples were positive for EHEC-Hly or E. coli O157. In combination of western blot results, bacterial isolation clinical symptoms and epidemiological investigation findings, it was reasonable to conclude that this cluster of patients with distinguish clinical symptoms was caused by E. coli O157:H7, which had never been reported in China. Hence serological methods with EHEC-Hly and E. coli O157 LPS are valuable for diagnosis of infections of E. coli O157:H7, when bacterial isolation is failed.

Adult ; Aged ; Antibodies, Bacterial ; blood ; Escherichia coli Infections ; complications ; immunology ; Escherichia coli O157 ; immunology ; Hemolysin Proteins ; immunology ; Hemolytic-Uremic Syndrome ; etiology ; immunology ; Humans ; Immunoglobulin G ; blood ; Immunoglobulin M ; blood ; Lipopolysaccharides ; immunology ; Middle Aged

Actinobacillus pleuropneumoniae (A. pleuropneumoniae), the causative agent of porcine contagious pleuropneumonia (PCP), is a significant pathogen of the world pig industry, vaccination is potentially an effective tool for the prevention of PCP. The purpose of present study was to enhance the immunogenicity of A. pleuropneumoniae live vaccine strain HB04C- (serovar 7), which was unable to express ApxIA, and to develop effective multivalent vaccines for the respiratory pathogens based on the attenuated A. pleuropneumoniae. We introduced a shuttle vector containing intact apxIA gene into HB04C-, generating HB04C2, an A. pleuropneumoniae serovar 7 live attenuated vaccine strain co-expressing ApxIA. Then we investigated the biological characteristics of HB04C2. We found that the shuttle vector expressing ApxIA was stable in HB04C2, and the growth ability of HB04C2 was not affected by the shuttle vector. We observed that HB04C2 elicited detectable antibodies against ApxIA and ApxIIA when it was administrated intratracheally as a live vaccine in pigs, and all immunized pigs were protected from heterologous virulent A. pleuropneumoniae (serovar 1) challenge. In conclusion, we demonstrated that A. pleuropneumoniae live vaccine could be used as a vector for expression of heterologous antigens.
Actinobacillus Infections ; prevention & control ; veterinary ; Actinobacillus pleuropneumoniae ; classification ; immunology ; Animals ; Bacterial Proteins ; biosynthesis ; genetics ; Bacterial Vaccines ; biosynthesis ; immunology ; Hemolysin Proteins ; biosynthesis ; genetics ; Pleuropneumonia ; microbiology ; prevention & control ; Swine ; Swine Diseases ; microbiology ; prevention & control ; Vaccines, Attenuated ; biosynthesis ; immunology

According to the GenBank sequences (GenBank Accession No. AF539467), one pair of primers was designed to amplify hly gene of Aeromonas hydrophila by PCR. After sequencing, homology analysis indicated that a DNA fragment of 1485 bp was amplified from isolated DNA from Aeromonas hydrophila, and it shared more than 99% homology in nucleotide sequence compared with other reference strains in GenBank. The gene was cloned in pET-28a vector to construct a recombinant plasmid pET-28a-hly, which was transformed into Escherichia coli BL21 (DE3), and the recombinant strain BL21(DE3)(pET-28a-hly) was obtained. The hemolysin was highly expressed when the recombinant strain BL21 (DE3) (pET-28a-hly) was induced by IPTG. The expressed protein was 56 kD as estimated by 15% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The immunogenicity of the expressed Hly protein was confirmed by Western blotting. Mice were immunized with inactivated whole bacteria vaccine and the genetic engineering vaccines showing promise that all these vaccines have a high protective ability. The results showed that the recombinant strain BL21 (DE3)(pET-28a-hly) could be candidate of hemolysin toxoid vaccine to provide protective immunity against diseases caused by Aeromonas hydrophila.
Aeromonas hydrophila ; genetics ; Animals ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Female ; Hemolysin Proteins ; genetics ; metabolism ; Immunization ; Mice ; Molecular Sequence Data ; Random Allocation ; Recombinant Proteins ; genetics ; immunology ; metabolism ; Sequence Homology ; Toxoids ; immunology ; Vaccines, Synthetic ; immunology

We previously induced protective immune response by oral immunization with yeast expressing the ApxIIA antigen. The ApxI antigen is also an important factor in the protection against Actinobacillus pleuropneumoniae serotype 5 infection; therefore, the protective immunity in mice following oral immunization with Saccharomyces cerevisiae expressing either ApxIA (group C) or ApxIIA (group D) alone or both (group E) was compared with that in two control groups (group A and B). The immunogenicity of the rApxIA antigen derived from the yeast was confirmed by a high survival rate and an ApxIA-specific IgG antibody response (p < 0.01). The highest systemic (IgG) and local (IgA) humoral immune responses to ApxIA and ApxIIA were detected in group E after the third immunization (p < 0.05). The levels of IL-1beta and IL-6 after challenge with an A. pleuropneumoniae field isolate did not change significantly in the vaccinated groups. The level of TNF-alpha increased in a time-dependent manner in group E but was not significantly different after the challenge. After the challenge, the mice in group E had a significantly lower infectious burden and a higher level of protection than the mice in the other groups (p < 0.05). The survival rate in each group was closely correlated to the immune response and histopathological observations in the lung following the challenge. These results suggested that immunity to the ApxIA antigen is required for optimal protection.
Actinobacillus Infections/prevention & control ; Actinobacillus pleuropneumoniae/genetics/*immunology ; Animals ; Antibodies, Bacterial/blood ; Bacterial Proteins/analysis/*immunology ; Cytokines/analysis/blood ; Disease Models, Animal ; Female ; Hemolysin Proteins/analysis/*immunology ; Immunoglobulin A/blood/immunology ; Intestines/immunology ; Lung/cytology/immunology ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins/*immunology ; Saccharomyces cerevisiae/*genetics/immunology ; Survival Analysis ; Time Factors ; Vaccination ; Vaccines, Synthetic/administration & dosage/*immunology

ApxI is one of the most important virulence factors of Actinobacillus pleuropneumoniae (APP). To study the immunogenicity of the ApxI, the complete coding sequence (3146bp) and its 5'-terminal 1140 bp fragment of the apxIA gene were separately cloned into the prokaryotic expression vector pET-28a, and expressed in the E. coli BL21 (DE3) with induction by IPTG. The expression products, rApxIA and rApxIAN, were present in a form of inclusion bodies and showed the same immunological reactivity as natural ApxI (nApxI) in Western-blot analysis. BALB/c mice were intraperitoneally immunized with the rApxIA, rApxIAN and nApxI respectively. The serum antibody levels of the rApxIAN immunized mice were significantly lower than those immunized with rApxIA or nApxI in an ApxI-specific ELISA, but serum neutralization test demonstrated that immunized mice with rApxIAN, rApxIA and nApxI could generate similar levels of antibodies neutralizing the hemolytic activity of the natural ApxI. The rApxIAN was able to elicite 80% protection rate against APP serovar 1 and 100% against serovar 2 when challenged at a dose of one LD50 after 2 weeks of boost immunization.
Actinobacillus Infections ; prevention & control ; Actinobacillus pleuropneumoniae ; genetics ; immunology ; Animals ; Antibodies ; blood ; Bacterial Proteins ; genetics ; immunology ; Bacterial Toxins ; genetics ; immunology ; Bacterial Vaccines ; immunology ; Cytotoxins ; genetics ; immunology ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Hemolysin Proteins ; genetics ; immunology ; Inclusion Bodies ; genetics ; immunology ; Mice ; Mice, Inbred BALB C ; Peptides ; genetics ; immunology ; Recombinant Proteins ; genetics ; immunology

Oral vaccination may be the most efficient way of inducing an immune response at the remote mucosal site through the common mucosal immune network. Antigenspecific secretory IgA (sIgA) is the major immunoglobulin type generally detected in the secretions of experimental animals following an effective oral immunization. Actinobacillus pleuropneumoniae causing disease in the lung of pig initially interacts, colonizes, and infects the host tissues at the mucosal surface of the respiratory tract. Also, importantly for A. pleuropneumoniae protection, the quantity of sIgA in the lung had merits associated with the mucosal immunity. However, there is no simple method to monitor the level of sIgA as an indicator for the induction of local immune responses by an oral vaccination in the target tissue. Therefore, the relationship between sIgA and IgG was analyzed to evaluate the induction of local immune responses by an oral immunization with Saccharomyces cerevisiae expressing the apxIA and apxIIA genes of A. pleuropneumoniae in this study. The correlation coefficient of determination (r2 x 100) for paired samples in both vaccinated and control groups showed a significant positive-relationship between IgG in sera and sIgA in the lung or intestine. These results indicated that IgG antibody titers in sera could be useful to indirectly predict local immune response, and sIgA, in the lung or intestine to evaluate the efficacy of an oral vaccination.
Actinobacillus pleuropneumoniae ; Administration, Oral ; Animals ; Antigens, Fungal/*immunology ; Bacterial Proteins/genetics/immunology ; Bacterial Vaccines/*immunology ; Disease Models, Animal ; Female ; Hemolysin Proteins ; Immunity, Mucosal/*immunology ; Immunoglobulin A, Secretory/*analysis ; Immunoglobulin G/*blood ; Intestine, Small/immunology ; Lung/immunology ; Mice ; Mice, Inbred BALB C ; Saccharomyces cerevisiae/*immunology

Acute toxicity and immunoprotection of Actinobacillus pleuropneumoniae (APP) ApxI toxin recombinant proteins (include crude inclusion bodies and refolded recombinant protein) were evaluated in mice, and compared with the natural ApxI extracted from culture supernatant of APP serotype 10. In the acute toxicity experiment, the three proteins were intraperitoneally injected into Kunming mice in a dose of 200microg per mouse. The body and organ weight, heamatological and biochemical indexes were examined at 24h, 7 days and 14 days post administration. There was no death after the intraperitoneal administration of the three proteins, and no significant change was found in the body weight, organ indexes, heamatological and biochemical indexes. To study their immunoprotection, the three proteins were emulsified with Freund's adjuvant respectively and vaccinated the mice twice with a 2-week of interval. Two weeks after the second vaccination, the mice were challenged intraperitoneally with a lethal dose of APP serotype 10 (1.09 x 10(8) cfu), and serums were examined by an ApxI-specific ELISA. The results revealed that the recombinant protein had a good immunogenicity and could induce protection immune reaction.
Actinobacillus Infections ; prevention & control ; Actinobacillus pleuropneumoniae ; genetics ; immunology ; metabolism ; Animals ; Bacterial Proteins ; genetics ; immunology ; Bacterial Vaccines ; genetics ; immunology ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Female ; Hemolysin Proteins ; genetics ; immunology ; Immunization ; Male ; Mice ; Random Allocation ; Recombinant Proteins ; genetics ; immunology ; Toxicity Tests, Acute