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.

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.

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.

Exposure to ozone reduces influenza disease severity and alters distribution of influenza viral antigens in murine lungs.

Exposure to ambient levels of ozone (0.5 ppm) was shown to alter the pathogenesis of respiratory infection after aerosol infection of mice with influenza A virus. A semiquantitative method for determination of the sites of virus replication by direct immunofluorescence indicated that exposure to ozone reduced the involvement of respiratory epithelium in the infectious process and resulted in a less widespread infection of the alveolar parenchyma. Furthermore, the ozone-mediated alteration in viral antigen distribution was consistent with significantly reduced influenza disease mortality and prolonged survival time, but only when the oxidant was present during the course of infection. Reduced disease severity in ozone-exposed animals appeared to be independent of peak pulmonary virus titers, pulmonary interferon titers, and pulmonary and serum-neutralizing antibody titers. These studies suggested that the distribution of influenza virus in the murine lung was a key factor in disease severity.

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.

Dynamics of B-lymphocytes in the lungs of mice exposed to aerosolized influenza virus.

Immunoglobulin-containing cells were revealed by immunofluorescence in lung sections from mice infected with influenza virus by the aerosol route. The numbers of immunoglobulin A (IgA)- and IgM-containing cells were increasing by day 3 of the infection, whereas IgG-containing cells appeared a few days later. The responding B-cell populations appeared in two principal locations: along major airways and in consolidated lesions within lung parenchyma. IgA-containing cells were the most numerous isotype, occurring predominantly in the lamina propria of the airways. IgG-containing cells were the least frequently encountered class along airways and appeared most often within consolidated lung lesions in clustered groupings. Cells staining for mu chain appeared along the airways and in lung lesions. The population of IgM-containing cells declined approximately 30 days after infection. Cells producing alpha and gamma chains were still numerous on day 46. Assays for virus-reactive antibodies in lung secretions were positive on day 8 of the infection. The IgM titers were the first to decline, but virus-binding antibodies for all classes were still present on day 33. The implications of immune responses in viral pneumonitis were considered.

Immunoglobulin E-containing cells in mouse lung following allergen inhalation and ozone exposure.

Cells containing immunoglobulin E (IgE) were enumerated and their location in mouse lungs was determined by direct immunofluorescence. Lungs were studied from mice that had been immunized with aerosolized ovalbumin as well as from normal mice and from mice that were exposed to ozone (0.5 or 0.8 ppm) prior to receiving aerosolized antigen. In addition, some mice were immunized intraperitoneally with ovalbumin precipitated in alum. IgE-containing cells were primarily airway-related in normal mice and in mice immunized by the intraperitoneal route. Lungs from aerosol-immunized, and aerosol-immunized ozone-exposed mice showed a more disseminated distribution of IgE-containing cells. Fluorescent cells were counted and numbers were expressed as total cells per square millimeter of lung tissue and as airway-associated cells per millimeter of airway. Total IgE cells increased 9.4-fold in mice that received aerosolized ovalbumin as compared to normal mice. When ozone exposure was added to the effects from aerosolized ovalbumin, the increase of IgE cells over normal was 34.2-fold. IgE cell counts correlated well with anaphylactic sensitivity to intravenous challenge with ovalbumin. The observed enhancement of allergic sensitization by ozone exposure has important implications for human health.

Studies on the enhancement of allergic lung sensitization by inhalation of ozone and sulfuric acid aerosol.

Air pollutants were found to enhance the allergic sensitization of mice to an inhaled antigen. Aerosolized ovalbumin was used to mimic the inhalation of an environmental allergen. In three experiments the antigenic contact was repeated at 4 to 7 times over a period of approximately a month. Groups of mice were intermittently exposed to ozone at 0.5 and 0.8 ppm, sulfuric acid aerosol (1 mg/m3), and a combination of the two air pollutants. Antigenically sensitized mice showed some evidence of atopic reactivity to the inhaled antigen, but the interpretation of these responses was difficult to evaluate by observation alone. Clear evidence of allergic sensitization was obtained by injecting the antigen intravenously and recording the instances of systemic anaphylaxis. Allergic mice demonstrated anaphylactic shock within a few minutes of the injection, and fatally shocked animals died within 20 to 40 min. Significant increases in the levels of sensitization were obtained in animals exposed to ozone and the combination of ozone and sulfuric acid aerosol.

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.

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.

Antibody responses and interferon titers in the respiratory tracts of mice after aerosolized exposure to influenza virus.

We studied the temporal appearance of immunoglobulins (immunoglobulins G1, G2, M, and A) and interferon in lung lavage fluids of mice after aerosol exposure to influenza virus in six animal groups in which mortality rates ranged from 0 to 24%. Immunoglobulin levels in the lung lavage fluids were markedly higher in mouse groups with higher mortality rates (16, 20, and 24%) than in those with low mortality rates (0, 2.5, and 7.5%). Analysis of serum albumin in the respiratory secretions as an index of edema indicated that increased immunoglobulin G levels during the early phase of infection were due to increased vascular permeability. The detection of virus-neutralizing antibodies and antibodies reactive with influenza virus antigens in the lavage fluids at 6 to 8 days postinfection suggested local immunoglobulin synthesis as a result of antigenic stimulation. Both systemic and local antibody productions contributed to immunoglobulin levels in the respiratory secretions after aerosolized influenza virus infection. Peak levels of interferon in the lavage fluids were reached before detection of significant levels of virus-neutralizing antibody in the serum or the lung lavage.

Immunofluorescence determination of the pathogenesis of infection with influenza virus in mice following exposure to aerosolized virus.

The pathogenesis of infection with influenza A virus in mice was studied by exposure of specific pathogen-free mice to aerosols of influenza virus and by monitoring of mortality, viral titers in lung homogenates, and presence of viral antigens in respiratory cells as determined by immunofluorescence. In two experiments with different death rates (100% and 43%), viral antigen accumulated in the epithelial cells lining the airways, in alveolar macrophages, in alveolar cells, and in visceral pleura. By enumeration of the number of airways, alveolar macrophages, and alveolar cells containing influenza viral antigens at different intervals after exposure to the viral aerosol, it was determined that viral replication occurred initially in the epithelial cells lining the airways and later extended to the alveolar macrophages and alveolar cells. This semiquantitative survey of the dynamics of influenza viral infection by aerosol indicated that the viral infection in mice was a descending process.

Pulmonary changes induced by low-level ozone: morphological observations.

This study defines the surface and ultrastructural changes which occur in mouse lungs during the early stages of continuous low-level ozone exposure. Swiss-Webster mice were exposed for 35 consecutive days to 0.5 ppm ozone, a level of oxidant gas which simulates levels occurring during an episode of severe smog in urban areas of the California south coast air basin. Groups of mice were killed on day 7, 21, or 35 of exposure and their lungs excised and examined by light microscopy, scanning electron microscopy, and transmission electron microscopy. Lung damage was most severe at the transition zone from terminal bronchiole to alveolar duct. This centriacinar lesion consisted of (1) increased numbers of macrophages within proximal alveoli of alveolar ducts, (2) clusters of type 2 pneumonocytes lining these proximal alveoli, (3) changes in surface characteristics of Clara cells, and (4) hyperplastic nodules of bronchiolar epithelium within terminal bronchioles. Inflammatory cell infiltrates were reduced in numbers at 35 days of exposure as compared to 7 days of exposure, but the hyperplastic bronchiolar epithelium persisted and, in fact, increased in severity as exposure length increased. Although inflammatory cell infiltrates were observed in proximal alveoli of alveolar ducts, the hyperplastic bronchiolar epithelial changes were observed throughout the lengths of terminal bronchioles examined. The variability in response to ozone insult of nonciliated cells in the terminal bronchiole of rats, mice, and monkeys is discussed.

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.

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.