Cell-mediated and humoral immune responses in mice during experimental infection with Mycoplasma pulmonis.

Cell-mediated and humoral immune responses in mice after challenge exposure with Mycoplasma pulmonis were investigated. The cell-mediated immune response was determined by means of the delayed-type footpad swelling and the humoral immune response by means of the indirect haemagglutination test. Delayed-type footpad swelling and serum antibody titres were detected at one week after the challenge exposure and persisted for 7 weeks until the end of the experiment. However, there was a poor correlation between the degree of delayed-type footpad swelling and that of serum antibody titre. Delayed-type footpad swelling in mice with gross pneumonic lesions was less than that of mice with no gross lesions. A weak negative linear correlation was observed between the delayed-type footpad swelling and the number of M. pulmonis isolated from lungs.

Enhancement of phagocytosis and bactericidal activity of neutrophils in miniature pigs by dihydroheptaprenol, a synthetic polyprenol derivative.

Dihydroheptaprenol (DHP), a synthetic polyprenol derivative, markedly stimulated the generation of peripheral blood neutrophils after intramuscular injection in miniature pigs. The generated neutrophils exhibited enhanced phagocytic activity against latex particles and also enhanced killing activity against Escherichia coli. The effective dose in miniature pigs (1.4 mg/kg) was markedly less than that required in mice (100 mg/kg). These results indicate that DHP induces resistance to some bacterial infections in pigs, suggesting the applicability of DHP for humans.

Passive hemagglutination test for detection of antibodies against Taylorella (Haemophilus) equigenitalis in sera of mares.

The passive hemagglutination (PHA) test was improved to enable the detection of antibodies to Taylorella (Haemophilus) equigenitalis in the sera of mares. Horse red blood cells (RBC) fixed with glutaraldehyde were compared with similarly treated RBC of a cow, pig and sheep for the PHA test. The horse RBC were superior to those of the other animals tested in detecting mares affected with contagious equine metritis (CEM). A PHA test using these cells as indicator and an antigen prepared from T. equigenitalis by sonication following treatment with hyaluronidase was the most satisfactory in terms of sensitivity and specificity. None of the 156 serum samples from clinically healthy mares without a history of contact with T. equigenitalis-infected stallions or mares showed PHA titers greater than 1:32 and only a few samples (7.1%) showed PHA titers of 1:32. Four of the 50 serum samples from mares affected with CEM showed PHA titers of 1:32, while most of the samples (92.0%) showed PHA titers greater than 1:32. The glutaraldehyde-fixed horse RBC sensitized with the antigen had the advantage of being reproducible for at least 7 months when preserved at 4 degrees C.

Cell-mediated and humoral immune response to non-viable Mycoplasma pulmonis in mice enhanced by cross-linked ricin.

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.

Humoral immune response to non-viable Mycoplasma pulmonis in mice enhanced by dextran sulfate.

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.

Prolonged excretion and failure of cross-protection between distinct serotypes of bovine rotavirus.

Four newborn calves were experimentally infected with two distinct serotypes of bovine rotavirus (BRV-1 and BRV-2). Initially, three colostrum-deprived calves were inoculated orally with either BRV-1 or BRV-2; all developed severe diarrhea and produced serotype-specific neutralizing antibodies. Fecal virus was first demonstrated by immunofluorescence the day after inoculation. The virus titers reached a maximum of 10(5.2)-10(6.6) fluorescent focus forming units g-1 of feces 2-5 days after inoculation and then decreased. Fecal virus was detected in low titers beyond 28 days after inoculation despite the development of serum neutralizing antibodies. One calf, which had acquired specific active immunity against BRV-1 following oral infection, was further infected orally with BRV-2 4 weeks later. The calf again manifested diarrhea, excreted BRV-2 and showed an increase in serum neutralizing antibody against BRV-2. These results indicated that calves infected with either BRV-1 or BRV-2 do not have cross-protection to infection with heterologous BRV, and that recurrence of the disease can occur. The possible mechanisms of the persistence of BRV in calves and its role in the epidemiology of this infection are discussed.

Cell-mediated and humoral immune response to Mycoplasma hyopneumoniae in pigs enhanced by dextran sulfate.

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.

Serotypes of bovine rotaviruses distinguished by serum neutralization.

Fourteen cytopathic bovine rotaviruses isolated from naturally affected calves were characterized serologically by serum neutralization. Antisera were prepared in guinea pigs against the three isolates and the Lincoln strain of bovine rotavirus. These four rotaviruses were segregated into at least two distinct serotypes (designated types 1 and 2) by serum neutralization. The remaining 11 isolates were also placed into two groups according to neutralization specificity by using the antisera against types 1 and 2 bovine rotavirus. When colostrum-deprived calves were experimentally inoculated with type 1 or 2 bovine rotavirus, respectively, the calves produced neutralizing antibodies against the inoculated serotype but not against the other serotype. Calf sera obtained from six herds with naturally occurring rotavirus infections possessed neutralizing antibodies against type 1 or 2 or both. Thus, distinct serotypes of bovine rotavirus were demonstrated by serum neutralization. The possible role of serotypes in the epidemiology of rotavirus infection among bovine species is discussed.

Primary isolation of cytopathic bovine rotaviruses on fetal rhesus monkey kidney cells.

Primary isolation of bovine rotaviruses was successfully performed on rolling cultures of MA104 cells following trypsin treatment of fecal samples and cells. Fifty-one fecal samples were obtained from 22 herds affected with naturally-occurring acute diarrhea in calves during a period of over two years. Rotavirus particles were demonstrated in only 10 fecal samples by electron microscopy. Fourteen cytopathic bovine rotaviruses were isolated from positive samples and could be serially cultivated on MA104 cells. The presence of virus was identified by specific immunofluorescence in infected cells. These data indicated that approximately 30% of the herds affected with acute diarrhea in their calves were associated with rotavirus infection.

Delayed hypersensitivity to nonviable Mycoplasma pulmonis in mice is enhanced by dextran sulfate.

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.

Improvement of complement fixation test antigen for the diagnosis of Mycoplasma hyopneumoniae infection.

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.

Rapid detection by counterimmunoelectrophoresis of antigens and antibodies in the sera of chickens infected with Leucocytozoon caulleryi.

The method of counterimmunoelectrophoresis was used to detect rapidly soluble antigens and antibodies in the sera of chickens infected with Leucocytozoon caulleryi. This method retained the specificity revealed by the Ouchterlony gel-diffusion technique, and induced no false positive reactions. Therefore, it was applicable to the serological diagnosis of chicken leucocytozoonosis, as well as the Ouchterlony technique which is used in Japan at present.

Suppression of immunoresponses to Haemophilus gallinarum with nonviable Mycoplasma gallisepticum in chickens.

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.