The immunologic reactivity of a lipopolysaccharide (LPS)-protein complex isolated from a potassium thiocyanate extract of a Pasteurella multocida (capsular type A and somatic type 3) strain was evaluated in mice. The LPS-protein complex provided 100% protection in mice against a challenge with the homologous strain. However, when the complex was fractionated into LPS and protein moieties by phenol-water treatment, both components lacked immunogenicity. The complex and extracted components were mitogenic for mouse B lymphocytes with the protein moiety the most active. Although immune serum against the LPS-protein complex protected mice against challenge thereby indicating a role for humoral immunity, the LPS-protein complex of P. multocida was also found to induce cell-mediated immunity. This cell-mediated immunity was demonstrated in mice immunized with the complex by: (1). mitogenic responses of T lymphocytes, (2). induction of delayed type hypersensitivity reaction in the hind footpads, and (3). enhanced resistance to challenge infection with Salmonella enteritidis.
Animals ; Antibodies, Bacterial/blood/immunology ; Bacterial Proteins/chemistry/*immunology ; Chemical Fractionation ; Hypersensitivity, Delayed ; Immune Sera/immunology ; Immunity, Cellular ; Immunization, Passive ; Lipopolysaccharides/chemistry/*immunology ; Lymphocyte Activation ; Mice ; Pasteurella Infections/immunology/*prevention & control ; Pasteurella multocida/*chemistry/immunology ; Salmonella Infections, Animal/immunology/prevention & control ; Salmonella enteritidis/growth & development/immunology ; Spleen/cytology/immunology/microbiology

OBJECTIVETo study the regulating effects of a novel CpG oligodeoxynuleotide and the synergistic effect of chitosan-nanoparticles (CNP) with CpG on immune responses of mice, which were used to develop a novel immunoadjuvant to boost immune response to conventional vaccines.

METHODSA novel CpG ODN containing 11 CpG motifs was synthesized and its bioactivities to stimulate the proliferation of lymphocytes of pig in vitro were detected. Then it was entrapped with CNP prepared in our laboratory by the method of ionic cross linkage, and immunized Kunming mice were co-inoculated with paratyphoid vaccine. The peripheral blood was collected weekly from the tail vein of inoculated mice to detect the contents of IgG, IgA, IgM, and specific antibody against salmonella as well as the levels of interleukin-2 (IL2), IL-4, and IL-6 by SABC-ELISA assay. The numbers of leucocytes, monocytes, granuloytes, and lymphocytes were calculated separately using the routine method. The experimental mice were orally challenged with virulent salmonella 35 days after inoculation.

RESULTSThis CpG ODN could remarkably provoke the proliferation of lymphocytes of pig in vitro in contrast with the control (P < 0.05). Compared with those of the control, immunoglobulins, including IgG, IgA, IgM, and specific antibodies to paratyphoid vaccine, increased significantly in sera from the CpG or CpG-CNP-vaccinated mice (P < 0.05). IL-2, IL-4, and IL-6 increased remarkably in sera from immunized mice (P < 0.05). The leucocytes, monocytes, granuloytes, and lymphocytes of the mice immunized with CpG or CpG-CNP were also increased in number (P < 0.05). After the challenge, these immunity values were elevated in the mice vaccinated with CpG or CpG-CNP. The immunized mice all survived, while the control mice fell ill with evident lesions with diffuse hemorrhage in stomach, small intestine, and peritoneum.

CONCLUSIONSCpG ODN entrapped with CNP is a promising effective immunoadjuvant for vaccination, which promotes humoral and cellular immune responses, enhances immunity and resistance against salmonella by co-administration with paratyphoid vaccine.

Adjuvants, Immunologic ; administration & dosage ; pharmacology ; Animals ; Antibodies, Bacterial ; blood ; Cell Proliferation ; Chitosan ; chemistry ; Drug Therapy, Combination ; Enzyme-Linked Immunosorbent Assay ; Immunoglobulin Isotypes ; blood ; Interleukins ; blood ; Lymphocyte Activation ; drug effects ; Lymphocytes ; cytology ; Mice ; Nanoparticles ; chemistry ; Oligodeoxyribonucleotides ; administration & dosage ; pharmacology ; Paratyphoid Fever ; immunology ; prevention & control ; Salmonella ; physiology ; Salmonella Infections, Animal ; immunology ; prevention & control ; Swine ; immunology ; Typhoid-Paratyphoid Vaccines ; immunology