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The enhancing effect of dextran sulfate (DS) on delayed hypersensitivity to nonviable Mycoplasma pulmonis in mice was evaluated by means of delayed footpad swelling. Mice pretreated subcutaneously with nonviable M. pulmonis and DS had significantly increased delayed footpad swelling when the mice were injected subcutaneously in the footpad with the same antigen 7 days later. Delayed footpad swelling was not detected in mice pretreated only with nonviable M. pulmonis or DS followed by footpad injection with nonviable M. pulmonis. Subcutaneous pretreatment in the scruff of the neck with nonviable M. pulmonis and DS was more effective in eliciting delayed footpad swelling than intravenous or intraperitoneal pretreatment. Delayed footpad swelling did not develop when mice were injected in the footpad with nonviable Mycoplasma hyopneumoniae, M. gallisepticum, M. dispar, or M. bovis on day 7 after pretreatment with M. pulmonis and DS. Injection of nonviable M. pulmonis in the footpad on day 3 after pretreatment with M. pulmonis and DS resulted in no significant footpad swelling, but injection in the footpad on days 7, 14, 21, and 28 resulted in significant footpad swelling. Delayed footpad swelling was transferred by intravenous injection with spleen cells of mice which were pretreated with nonviable M. pulmonis and DS into nontreated recipient mice. Intravenous injection with anti-mouse thymus cell rabbit serum into the mice which were previously pretreated with nonviable M. pulmonis and DS reduced the delayed footpad swelling significantly.

Several attempts were made to develop a complement fixation test antigen which is useful and practicable for the diagnosis of mycoplasmal pneumonia of swine. For the preparation of antigen, Mycoplasma hyopneumoniae was grown in broth medium containing horse serum for 14 days by shaking culture. Anticomplementary activity of antigen was eliminated by addition of complement and heating at 56 degrees C for 30 minutes. Storage of this antigen at 4 degrees C or at room temperature for 12 months had no significant effect on antigen titer. In the complement fixation test with this antigen, complement-fixing antibodies could be detected up to 41 weeks after experimental inoculation by KIO4 treatment of serum. They were demonstrated in 81.4% of 296 sera collected at slaughterhouses in five prefectures.

The effect of dextran sulfate (DS), known to be cytotoxic to macrophages, on the cell-mediated and humoral immune response to nonviable Mycoplasma hyopneumoniae in pigs was investigated. The cell-mediated immune response was determined by means of lymphocyte transformation a test, using uptake of [3H]thymidine in a microculture system and the humoral immune response by means of a microplate complement-fixation test. Peripheral blood lymphocytes from pigs vaccinated with nonviable M hyopneumoniae and DS incorporated substantially more [3H]thymidine than did those from pigs given Mycoplasma or DS alone. The transformation of lymphocytes from M hyopneumoniae-DS vaccinated pigs was enhanced when M hyopneumoniae cells used in the assay system were heated at 60 C for 30 minutes. Similarly prepared M flocculare and M hyorhinis cells also stimulated lymphocytes from M hyopneumoniae-DS vaccinated pigs, but not nearly as great as when M hyopneumoniae cells were used. The humoral antibody response and the cell-mediated immune response to nonviable M hyopneumoniae was markedly enhanced by DS. Pigs were vaccinated with nonviable M hyopneumoniae and/or DS 4 times and challenge exposed intratracheally with viable M hyopneumoniae. Pigs vaccinated with M hyopneumoniae and DS had less severe pneumonia than did nonvaccinated pigs.

The suppressive effect of Mycoplasma gallisepticum (MG) on Haemophilus gallinarum (HG) immune response was shown. Antibody response to HG was highly suppressed when chickens were inoculated intramuscularly with HG-MG combined bacterin. Findings were similar in chickens injected intramuscularly with HG and MG bacterin separately at adjacent sites. No immunosuppressive effect was recognized when injections with HG and MG bacterins were in the left and right thigh muscles, respectively, or from intravenous inoculation with the combined bacterin. Nor did HG-Mycoplasma synoviae (MS) combined bacterin injected intramuscularly evidence immunosuppression. Recovery rate of HG and clinical symptoms were more evident in chickens with suppression of antibody responses than in chickens without suppression.

The enhancing effect of cross-linked ricin (CL-ricin) on the cell-mediated and humoral immune response of mice to non-viable Mycoplasma pulmonis was studied. The cell-mediated immune response was evaluated by means of the delayed-type footpad swelling, and the humoral immune response by means of the indirect hemagglutination test. Mice pre-treated subcutaneously with non-viable M. pulmonis and CL-ricin showed significantly increased delayed-type footpad swelling when they were injected in the footpad with the same antigen 7 days later. Delayed-type footpad swelling was not detected in mice pre-treated only with non-viable M. pulmonis or CL-ricin followed by footpad injection with non-viable M. pulmonis. Injection of non-viable M. pulmonis in the footpad on Days 3, 7, 14, 21, 28, 35 and 42 after pre-treatment with non-viable M. pulmonis and CL-ricin resulted in significant footpad swelling. Delayed-type footpad swelling was transferred by intravenous injection of spleen cells from mice which had been pre-treated 7 days previously with non-viable M. pulmonis and CL-ricin into non-treated recipient mice. Intravenous injection of anti-mouse thymus cell serum into mice previously pre-treated with non-viable M. pulmonis and CL-ricin reduced the delayed-type footpad swelling significantly. Mice pre-treated subcutaneously with non-viable M. pulmonis and CL-ricin showed a marked increase in serum antibody titers compared with those that received non-viable M. pulmonis alone.

The enhancing effect of dextran sulfate on the humoral immune response to nonviable Mycoplasma pulmonis in mice was evaluated by means of the indirect haemagglutination test. The serum antibody titres in mice immunized subcutaneously with a mixture of non-viable M. pulmonis and dextran sulfate were greater and persisted longer than those in mice immunized with non-viable M. pulmonis alone. DEAE-dextran also enhanced the humoral immune response to non-viable M. pulmonis in mice.