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Genetically susceptible BALB/c mice were immunized i.m. with DNA for one or two Mycoplasma pulmonis Ags (A7-1, A8-1) beginning either 1 wk before (vaccination) or 1 wk after (treatment) intranasal infection with 5 x 10(4) CFU virulent M. pulmonis organisms. Immunization of mice by this method induced both humoral and cellular immunity to M. pulmonis, largely prevented infection (vaccination), and cleared an ongoing pneumonia over time (treatment). Only one Ag gene was required. Thus, DNA immunization is a potential treatment for infections and may be useful in instances when drug therapy may not be available or effective.

The induction of an immune response against a foreign protein usually requires purification of that protein, which is injected into animals. The isolation of pure protein is time consuming and costly. Recently, a technique called biolistic transformation (biological ballistic system) microparticle injection, gene gun, or particle bombardment was developed. The basic idea is that DNA or biological material coated onto heavy tungsten or gold particles is shot into target cells or animals. We have vaccinated mice by introducing the gene (Mycoplasma pulmonis DNA or a specific fragment) encoding a protein recognized by a protective monoclonal antibody directly into the skin or muscle of mice by two methods: (i) using a hand-held form of the biolistic system that can propel DNA-coated gold microprojectiles (2 micrograms of DNA) directly into the skin; (ii) using a conventional intramuscular injection of the DNA (100 micrograms) into quadricep muscles of transfected mice. HeLa cells were transfected in vitro by the gene gun or by the liposomal delivery system. Indirect immuno-fluorescent antibody (IFA) assay of culture cells indicated that both methods could be successful. Production of antibody and cell-mediated immunity against M.pulmonis were monitored by assaying serum IFA and enzyme-linked immunosorbent assay (ELISA), and delayed type hypersensitivity. In addition, macrophage migration inhibition and lymphocyte transformation to antigen in spleen cells were also tested. Both delivery systems induced humoral and cellular immunity, and vaccinated the mice against infection. Genetic immunization by using the gene gun saves time, money, and labor; moreover, this general method is also applicable to gene therapy.

Results of competitive binding studies with radiolabeled and unlabeled Mycoplasma pulmonis and rat tracheal explant cultures indicated no effect of trypsin treatment on the ability of M. pulmonis to bind to explants. A trypsin-resistant 46-kDa membrane protein, which binds isolated rat tracheal epithelial cells in culture, was purified and used to produce specific antibodies that block adhesion of mycoplasmas to tracheal explants. These results suggest that M. pulmonis adheres to rat tracheal epithelium via a trypsin-resistant 46-kDa protein.

A construct of the Mycoplasma pulmonis (MP) genomic library, using randomly sheared DNA, was cloned in lambda gt11 and transfected into C600 Escherichia coli organisms. Clones of E. coli expressing a fusion protein reactive with anti-MP and monospecific serum were transferred orally or intravenously into Balb/c mice. Expression of the fusion protein was induced by adding isopropyl-beta-D-thiogalactopyranoside to the drinking water. This vaccination protocol led to local and systemic antibody formation, to generation of immune lymphocytes and to protection against large numbers of virulent MP organisms. This approach might be generally successful in preventing infectious disease.

Monoclonal antibodies (mAbs) were produced against Mycoplasma pulmonis (MP); some were highly protective in the treatment of experimental infections of BALB/c mice. The mAbs inhibited MP growth in vitro and prevented the attachment of MP to fibroblasts or to red blood cells. Three separate mAbs recognized 54-76% of 54 clinical isolates of MP, and the three together detected all 54 isolates. We used the mAbs to purify the antigens by affinity column chromatography. The purified antigen used to vaccinate mice and to immunize rabbits produced antibodies capable of significant growth inhibition in sera and tracheolung lavage fluids. The vaccinated mice were challenged with various doses of a highly virulent T2 strain of MP. Assays for viable MP organisms and for histopathological changes in the lungs of infected mice indicated that mice were protected if the challenge dose was 10(3)-10(5), but not 10(7), c.f.u. The sera of immunized rabbits were used to passively transfer immunity to mice. The sera provided complete protection against 1 x 10(6) c.f.u. T2 MP. We conclude that MP antigens purified by this protocol can provide a safe vaccine against this disease, at least in mice.

Temperature-sensitive mutant vaccines protect rats against Mycoplasma pulmonis infection. The role of the humoral or cellular immune response in resistance to mycoplasma infection was investigated by adoptive-transfer experiments. Spleen cells from Lewis rats vaccinated but not challenged with wild-type organisms (vaccinated) and spleen cells from rats vaccinated (or not) and challenged were effective in preventing syngeneic recipients from developing respiratory disease. There was also a significant reduction in the incidence and number of challenging organisms in the respiratory system. In contrast, sera from the same donors had no detectable effect on the number of mycoplasmas recovered or on lesion development in the respiratory tract. We conclude that cellular immunity rather than humoral immunity generated in vaccinated rats confers protection against subsequent infection.

Infection of C57BL/6J mice with Mycoplasma pulmonis (MP) enhanced NK cell function 3-7 days later, as detected by in vitro and in vivo assays. Moreover, spleen and lung cells of acutely infected C57BL/6J mice inhibited MP growth in vitro. The effectors were eliminated by treatment with anti-NK antibody in vivo and anti-asialo GM1 serum or anti-3A4 antibody plus complement in vitro. Clearance of viable and radiolabeled MP from the lungs was also enhanced in acutely infected mice. Acutely infected mice with severe combined immunodeficiency (SCID) eliminated viable MP faster than did uninfected mice. Antibodies to interferon-gamma (IFN-gamma) impaired clearance of MP from the lungs of SCID mice and decreased their survival times. Activated NK cells can function in resistance to early stages of infection with MP. NK cells directly inhibit MP with secrete IFN-gamma, which may activate macrophages or inhibit the growth of MP or both.

Chalquest and Hayflick media with and without ammonium reineckate were compared for isolation of Mycoplasma pulmonis from the nasopharyngeal ducts, tracheobronchial trees, and middle ears of 66 naturally infected rats. The results show that 92% (366 of 396) of the samples were positive for M. pulmonis in Chalquest medium with and without ammonium reineckate and 66% (260 of 396) were positive in Hayflick medium with and without ammonium reineckate (P less than 0.001). An enhancing effect of ammonium reineckate on M. pulmonis isolation was observed only in Hayflick medium; the isolation rate was 76% (151 of 198) in Hayflick medium with ammonium reineckate as compared with 55% (109 of 198) in Hayflick medium without ammonium reineckate (P less than 0.03). No significant differences in isolation rates were observed between Chalquest medium with and without ammonium reineckate. The mean growth time of M. pulmonis on Chalquest medium was 3.4 days as compared with 5.1 days in Hayflick medium, indicating that M. pulmonis can be detected earlier on Chalquest medium than on Hayflick medium. These data indicate that Chalquest medium is superior to Hayflick medium for M. pulmonis isolation from rats.