Surface markers on bovine fetal lymphocytes and immunoglobulin synthesis in a congenital infection related to epizootic bovine abortion.

Immunological parameters were studied among 23 late-term bovine fetuses. Epizootic bovine abortion (EBA) disease was induced in fetuses by feeding Ornithodoros coriaceus ticks on pregnant heifers. A spirochaete-like microorganism was detected in the blood of diseased fetuses and in inapparent natural infections in some abattoir-collected fetuses. Fetuses were classified according to stages of disease: EBA diseased (n = 10), EBA infected (n = 7) and normal (n = 6). Using flow cytometry, the presence of surface immunoglobulins (sIg) and peanut agglutinin (PNA) receptors were used to detect B and T lymphocytes, respectively. In peripheral blood of normal fetuses, most lymphocytes were identified as T or B cells, whereas about 20 per cent of lymphocytes in EBA diseased fetuses did not reveal the sIg or PNA receptor markers (null cells). Size and shape analyses by flow cytometry detected a population of enlarged lymphocytes in the EBA diseased fetuses. The numbers of cells bearing determinants reactive with monoclonal antibodies specific for bovine T cells (B26A and B29A) and B cells (TH21A) were considerably less than those expressing the PNA receptor and sIg. These results suggested that the monoclonal antibodies were binding to differentiation antigens which were not consistently expressed on the fetal cells. Radio-immunodiffusion was used to measure bovine IgM, IgG1 and IgG2 in fetal serum. The quantities of immunoglobulins were markedly increased in animals infected with the spirochaete-like organism (groups 1 and 2) and were assumed to result from fetal antibody synthesis.

Lymphocyte blastogenesis and cellular cytotoxicity in a congenital infection of bovine fetuses related to epizootic bovine abortion.

The disease referred to as epizootic bovine abortion (EBA) was experimentally induced in bovine fetuses. Dark-field microscopy was used to detect congenital infection with an unclassified spirochaete-like organism. Some of the fetuses collected at abattoirs were also found to be naturally infected with a morphologically similar microorganism. Blood counts and organ weights were correlated with the presence of the microorganism. Lymphocyte blastogenesis increased, the result of in vivo stimulation among the infected fetuses. Phytomitogens (phytohaemagglutinin, concanavalin A and lipopolysaccharide) also stimulated greater responses in infected fetuses when compared to results in normal fetuses. Cellular cytotoxicity was examined by the single cell assay and results indicated that there were fewer cytotoxic lymphocytes among the diseased fetuses. The infected abattoir-collected specimens were obtained from clinically normal adult cattle, and the immunological changes in these fetuses were closely characterised with those of the EBA diseased fetuses. These naturally infected fetuses showed signs of a mild infectious disease.

A procedure for pulmonary lavage in mice.

A method for the pulmonary lavage of mice is described. The procedure includes exsanguination of anesthetized mice by severting the renal artery, inserting a tracheal catheter in situ, and repeatedly injecting and aspirating 0.9% sodium chloride solution. Protein was recovered from the cell-free lavage fluid even after a given mouse was lavaged several times. The major part of the protein, however, was obtained with 1-ml washes repeated 3 times. Approximately 0.563 mg of protein was recovered by the procedure from a 29-g mouse. Four lavages per mouse yielded approximately 2.9 x 106 free cells.

Immunogenic collagen in induced ascitic fluid: concurrent preparation of anti-murine immunoglobulin E.

Antiserum to murine immunoglobulin (Ig) E was produced by inoculation of goats with a pool of partially purified IgE from serum and adjuvant-induced ascitic fluid. Antibodies to collagen were found to be present in the antiserum when the latter was conjugated with fluorescein isothiocyanate and applied to mouse pulmonic tissue. The intense connective tissue fluorescence was eliminated following absorption with mouse collagen. Immunogenic collagen components were presumed to arise in ascitic fluid as a consequence of the adjuvant-induced inflammation. Ascitic fluid is commonly used when large volumes of serum proteins are collected from small mammals. It is suggested that ascitic fluid may not be an ideal antigen source when antiserum is to be used for immunofluorescence studies on tissue.

Histopathologic changes in bovine fetuses after repeated reintroduction of a spirochete-like agent into pregnant heifers: association with epizootic bovine abortion.

A spirochete-like organism was found in the plasma of bovine fetuses affected with epizootic bovine abortion (EBA). The spirochete-like organism was frequently found in abattoir-collected fetuses as an inapparent infection, and EBA was found in cattle on foothill rangeland where the vector tick Ornithodorus coriaceus could repeatedly reintroduce the infectious agent into pregnant cattle (superinfection). Epizootic bovine abortion resembled a naturally acquired superinfection in circumstances where the agent was frequently present in the environment under conditions favoring transmission. Therefore, to determine whether fetal lesions could be experimentally induced in utero, spirochete-like organisms collected from clinically normal fetuses at an abattoir were inoculated IV and subcutaneously into 2 pregnant heifers 5 times over a 4-month period to mimic repeated tick transmission in the field. Macroscopic and microscopic examinations of tissues from 2 cesarean-collected fetuses and from 3 calves born at term with the naturally acquired spirochete infection indicated that the calves had evidence of an infection that caused morphologic changes compatible with immunologic stimulation and mild reticuloendothelial hyperplasia. Compared with findings in the calves, lesions in the superinfected fetuses were more severe, and the lesion distribution in various organs was more extensive. The spirochete-like organism appeared to be a mild pathogen because of its persistence in the host. Clinical disease from the infection may only develop with repeated superinfections. Therefore, a relationship between this microorganism and EBA is probable.

Congenital spirochetosis in calves: association with epizootic bovine abortion.

Congenital spirochetosis was encountered as a newly recognized infection of cattle. The spirochete was seen in blood of fetuses with lesions of epizootic bovine abortion. A spirochete with morphologic features similar to those found in the fetuses was detected in Ornithodoros coriaceus ticks. Ticks collected from rangelands were allowed to feed on cows that then produced epizootic bovine abortion-affected fetuses, and the fetuses had spirochetosis. Inapparent spirochetosis also was found in fetuses in clinically normal cattle sent to slaughter. Only a few lesions were seen in abattoir-collected fetuses. Fetal spirochetosis was common in the bovine population studied, and it appeared that infection may be limited only by the availability of the tick vector.

Immunological alterations in the lungs of mice following ozone exposure: changes in immunoglobulin levels and antibody-containing cells.

Ozone was added to the air of the environmental chambers containing specific pathogen-free mice. At levels of 0.5 and 0.8 ppm the oxidant was seen to have inflammatory effects, as shown by rising serum albumin levels in lung lavage fluid. Fluorescein conjugated anti-heavy chain sera were used to detect cells containing IgM, IgG, and IgA in measured lung areas termed Pulmonary Units. Antigenic stimuli occurred along the airways, with significant increases of IgA-containing cells in the bronchus-associated lymphoid tissue. The numbers of IgM- and IgG-containing cells did not increase. Immunodiffusion analyses for immunoglobulins in lung lavage fluid indicated increases of IgG1, IgG2, and IgA in lung secretions. The calculation of changing Ig/Alb ratios suggested that the IgA present was largely the result of local synthesis, while IgG molecules were mainly of serum origin. Possible sources of antigenic stimuli to ozone-exposed lungs are discussed.

Effects of antithymocyte sera and antimacrophage sera on cell-mediated immune reactions in Listeria-infected mice.

The cell-mediated immune responses of allograft rejection, delayed hypersensitivity, and resistance to Listeria monocytogenes were suppressed by injections of antithymocyte serum (ATS), but the immune responses were not significantly altered by antimacrophage serum (AMS) or normal rabbit serum (NRS). Antisera were prepared in rabbits against purified mouse thymocytes and purified peritoneal macrophages. When mice were injected with ATS near the time of skin grafting, allografts survived significantly longer. Similar administration of AMS or NRS failed to alter the course of graft rejection. Decreased footpad swelling indicated the suppression of delayed hypersensitivity in mice injected with ATS 6 days after a sublethal inoculation of Listeria cells. None of the serum treatments affected the ultimate survival of mice infected with a small number of bacteria. Either ATS or NRS was injected into immunized mice 1 day before and 2 days after a challenge inoculation of Listeria cells. Pronounced suppression of delayed hypersensitivity was found in the ATS-treated groups, along with extensive mortalities that reached 100% in the group receiving the largest dose of ATS. All control animals survived and demonstrated strong delayed hypersensitivity reactions. Antimacrophage serum had no significant effect on the three mechanisms of cell-mediated immunity that were tested. The lymphoid cells which mediate delayed hypersensitivity, antimicrobial cellular immunity, and allograft rejection possess antigenic determinants in common with thymocytes.

Effects of antithymocyte and antimacrophage sera on the survival of mice infected with Listeria monocytogenes.

Antisera prepared in rabbits against purified mouse thymocytes (antithymocyte serum; ATS) and peritoneal macrophages (antimacrophage serum; AMS) were injected intraperitoneally into Balb/c mice infected with the bacterium Listeria monocytogenes. When administered near the initiation of infection, the ATS significantly decreased the survival time of the animals and increased the mortality rate. When ATS was administered 6 days after a sublethal dose of L. monocytogenes had been inoculated, an overt disease did not evolve. ATS that significantly potentiated primary listeriosis also had high cytotoxicity titers for thymocytes and lymphoid cells from the peritoneal cavity. Although cytotoxic activity against peritoneal macrophages could be demonstrated in lower dilutions of the ATS, this activity did not appear to correlate with the effects of the sera on listeriosis. The injection of AMS did not enhance the infectious process. In some trials more deaths occurred among mice receiving normal rabbit serum than those receiving AMS. All of the AMS had cytotoxic titers against peritoneal macrophages, and the sera were usually inactive against thymocytes and peritoneal lymphoid cells. Listeria was isolated from fatally infected mice with nearly equal success in all of the serum-treated groups, and the serum treatments did not appear to alter the pattern of gross lesions. The afferent limb of the immune response was markedly affected by the presence of antibodies to lymphocytes. However, antibodies reacting with macrophages did not demonstrably enhance the Listeria process, which depends upon cellular immunity as the principal means of acquired host defense.

Cellular immunity of mice infected with Listeria monocytogenes in diffusion chambers.

The importance of bringing live bacteria into intimate contact with macrophages as a prerequisite for establishing cellular immunity was investigated. The bacterium Listeria monocytogenes was shown to replicate and survive in diffusion chambers implanted in the peritoneal cavities of mice. Humoral substances accruing from host responses to diffusing soluble antigens of the microorganism were unable to inactivate the bacteria. The resistance of mice immunized by subcutaneous inoculation of the live organism always exceeded the resistance of mice with Listeria diffusion chamber implants. Animals with sham diffusion chambers were more resistant to a challenge by L. monocytogenes than were normal mice. Host resistance was not significantly different between Listeria diffusion chamber implant groups and sham diffusion chamber implant groups. The results suggested that direct involvement of macrophages with the parasite is necessary to achieve cellular immunity.

The effects of ozone on the respiratory epithelium of mice II. Ultrastructural alterations.

Ultrastructural alterations in the tracheal and bronchial epithelium of mice exposed to 0.8 ppm ozone for varying periods of time were examined with scanning electron microscopy. The lesions were apparent in the ciliated cells. Examination of tissue from control mice showed that the ciliated cells were arranged in groups and the cilia were uniform in length. After six days of exposure to ozone, shortened cilia were occasionally observed by day 10, more pronounced changes were observed. Cilia were either absent or became short and blunt. The lesions observed after 20 days in ozone were similar to those seen on day 10. After ozone-exposed mice had been returned to ambient air for 10 days, ciliary regeneration occurred and, the major airways had a surface appearance approaching the normal state.

Mutation of Listeria monocytogenes After Prolonged In Vivo Survival in Diffusion Chambers.

Listeria monocytogenes remained viable when confined in diffusion chambers in the peritoneal cavities of mice. Bacterial survival was associated with mutations.

Relationship of antimicrobial cellular immunity to delayed hypersensitivity in Listeriosis.

The relationship of antimicrobial cellular immunity to delayed hypersensitivity (DH) was studied in mice antigenically stimulated by living Listeria monocytogenes confined to diffusion chambers in peritoneal cavities or by subcutaneous inoculation of sublethal doses of the organism. Mice showed DH reactions when tested 6 days after inoculation, and reactions were positive for at least 90 days in some mice. DH also became established when the mice were stimulated by antigens diffusing from peritoneal chambers containing viable Listeria. Mice were categorized as DH positive or DH negative if they developed more or less than a 5% increase in foot volume 24 h after the injection of Listeria antigen. Some antigenically stimulated mice did not elicit the DH reaction. Consequently, the animals were arranged as immunized groups (DH positive and DH negative) and Listeria chamber implant groups (DH positive and DH negative). When challenged with L. monocytogenes, all four groups were significantly resistant as compared with controls. Thus, the in vivo tests for immunity and DH did not show direct correlation. The results suggested that antimicrobial cellular immunity can occur as a phenomenon independent of DH. Evidence for antimicrobial cellular immunity as the principle mechanism of resistance in murine listeriosis is discussed with consideration for possible heterogeneity of function by thymus-derived lymphocytes.

Enhancement of allergic lung sensitization in mice by ozone inhalation.

Inhaled ozone was found to exert an enhancing effect for allergic lung sensitization when mice contracted an aerosolized allergen. The animals were exposed to ozone concentrations of 0.24, 0.16, 0.13, and 0.10 ppm. After 4 days of continuous ozone exposure, the mice had allergen contact from an aerosolized solution of ovalbumin. The animals were then maintained in ambient air for several days before the cycle of ozone and aerosolized allergen was repeated over four allergen contact cycles. Mice were rested in ambient air for a week after the last allergen contact, and they were then tested for allergic sensitization by the intravenous injection of 2 mg of ovalbumin to induce anaphylactic shock in allergic individuals. The control groups of mice were maintained in ambient air throughout the experiment, but they experienced identical allergen contact with the ozone-exposed mice. The phenomenon of allergic enhancement from ozone inhalation was detected at 0.24, 0.16, and 0.13 ppm of ozone. The enhancing effect disappeared at 0.10 ppm of ozone. The study indicated a potential for increasing the number of allergically sensitized individuals when various allergens are inhaled during periods of high ozone exposure with the consequent adverse changes on respiratory membranes. The significance to human health of the allergic enhancement phenomenon by ozone needs investigation.

The effects of ozone on the respiratory epithelium and alveolar macrophages of mice. I. Interferon production.

The effects of 0.8 ppm ozone on the capacity of the tracheal epithelium and alveolar macrophages of mice to produce interferon in vitro was studied. Exposure of mice to ozone for a period of 11 days or more affected the capacity of the tracheal epithelial cells in vitro to produce interferon. The inability of the tracheal epithelium in vitro to produce interferon was not due to the inhibition in the release of intracellular interferon but to an inhibition in the production of interferon. There was a complete recovery of the ability of tracheal epithelium to respond to interferon inducers after the mice were returned to ambient air 24 days post ozone exposure. However, ozone did not seem to have any affect on the capacity of the alveolar macrophages to produce interferon in vitro.

Immunochemical analysis of serum-related proteins in the respiratory tract secretions of normal mice.

Normal mouse lung lavage fluid was analyzed for its content of serum-related proteins by the methods of gel filtration, immunoelectrophoresis, and double immunodiffusion. Lavage samples were collected from exsanguinated mice using three repeated infusions of 0.9% sodium chloride solution. Cells were removed, and the lavage fluid was concentrated. Approximately 0.23 mg of protein per mouse was present in the concentrated solution. Albumin, transferrin, and immunoglobulin G (IgG) were among the proteins in greatest concentration. Albumin, IgG1, IgG2, IgA, and IgM were quantitated by single radial diffusion. Although immunoglobulins could be synthesized locally, albumin and transferrin were assumed to come from plasma. The albumin content was used to estimate the amount of transudated plasma as 0.0027 ml per mouse. The IgA levels were low with an IgA/IgG1 ratio of 0.58 and an IgA/IgG2 ratio of 0.43. IgM was not detected. Goat anti-lung lavage serum revealed the presence of additional lung-related proteins.

Activity of macrophage and neutrophil cellular fractions from normal and immune sheep against Listeria monocytogenes.

Cellular immunity to Listeria monocytogenes infection was studied by assaying for antibacterial activity in fractions of leukocytes collected from the peritoneal cavity, lungs, and mammary glands of immunized sheep. The cells were collected in populations that were largely either macrophages or neutrophils. Mechanically disrupted cells were divided into nuclear, lysosomal, and supernatant fluid fractions and then subjected to freezing and thawing. Comparison with similarly treated rabbit cells showed that greater fragility exists in the lysosomes of sheep cells, as indicated by the amount of acid phosphatase activity released. Inhibition of bacterial growth was assayed in a broth medium at pH 4.6. As expected, nuclear and lysosomal fractions from neutrophils were inhibitory. Some antibacterial activity was found in nuclear fractions of macrophages. The lysosomes of macrophages collected from the peritoneal cavity and the mammary gland did not inhibit the growth of L. monocytogenes. Peritoneal macrophages were allowed to interact with sensitized lymphocytes and an avirulent strain of L. monocytogenes for 4 hr prior to disruption and fractionation, but antibacterial activity was not detected. Pulmonary alveolar macrophages from 5 out of 16 sheep contained Listeria inhibitory activity in their lysosomes. The mechanism was inhibitory but not bactericidal.