Lack of airborne spread of infection by Legionella pneumophila among guinea pigs.

Many investigators find no spread of Legionnaires disease from person to person. The present study examined the question of airborne transmission of infection by Legionella pneumophila serogroup 1 from guinea pigs inoculated nasally with the agent to healthy guinea pigs. The nasal inoculation produced confluent peribronchiolar pneumonia similar to the pulmonary lesions observed in humans, but by techniques of clinical observation, serology, culture, and pathology, there was no evidence of airborne spread of infection from 26 inoculated guinea pigs to 64 uninoculated guinea pigs. The results, compatible with epidemiological studies of Legionnaires disease that fail to demonstrate airborne person-to-person transmission of the illness in humans, are useful for scientists who work with animal models of Legionnaires disease.     

In vitro responses of guinea pig peritoneal macrophages to Legionella pneumophila.

Transmission and scanning electron microscopy were used to study the phagocytosis of virulent and avirulent strains of Legionella pneumophila. The interaction between L. pneumophila and peritoneal macrophages from normal guinea pigs or from animals that had survived infection was studied. The virulent strains survived and proliferated within the phagocyte after ingestion by either type of macrophage, whereas the avirulent strain of bacteria was killed by normal macrophages. Although the addition of immune serum enhanced phagocytosis, the outcome was the same as with normal serum.     

Virulence conversion of Legionella pneumophila serogroup 1 by passage in guinea pigs and embryonated eggs.

When virulent Legionella pneumophila is passaged on supplemented Mueller-Hinton agar, it remains virulent for guinea pigs and embryonated hen eggs for two passages. However, by the fifth passage the cultures become avirulent for guinea pigs. Flagella were not produced by L. pneumophila on the first passage on supplemented Mueller-Hinton agar. In contrast, 12 passages on charcoal-yeast extract agar did not result in the reduction of virulence or the loss of flagella of L. pneumophila. Growth in supplemented yeast extract broth or on Norit-A-filtered supplemented yeast extract agar also did not result in a reduction of the virulence of L. pneumophila. However, L. pneumophila did not produce flagella when grown on these two media. Thus, it appears that the production of flagella is not required for the virulence of L. pneumophila when administered by the intraperitoneal route of infection. A virulent flagellated form of L. pneumophila was recovered by passing an avirulent form six times in guinea pigs. When avirulent L. pneumophila was passaged 12 times in embryonated eggs, a nonflagellated form of the bacterium was recovered which had an increased virulence for guinea pigs and embryonated eggs. However, virulent forms were not recovered by passage of avirulent forms on commonly used laboratory media. These results support the suggestion that a suitable host is required for the selection of the virulent form of L. pneumophila from avirulent cultures.     

Lymphoid cell blastogenesis as an in vitro indicator of cellular immunity to Legionella pneumophila antigens.

The lymphocyte blastogenic transformation assay was adapted to study responsiveness of lymphoid cells from animals and humans to Legionella pneumophila antigens in vitro. Spleen cells from guinea pigs after active immunization with Legionella vaccine, but not from normal animals, responded by blast cell transformation when stimulated in vitro with killed Legionella whole-cell vaccine, sonic extracts thereof, or a purified somatic antigen. The response was dose dependent. Similar lymphocyte blastogenesis occurred with spleen cells from mice sensitized to Legionella by sublethal infection with the bacteria. In addition, blastogenesis occurred with peripheral blood leukocytes from human volunteers tested in vitro with whole-cell vaccine, sonic extracts, or purified somatic antigen. Maximum responsiveness generally occurred 4 to 5 days after stimulation of human peripheral blood leukocytes, but a day or two earlier with spleen cells from normal or sensitized mice. Guinea pig spleen cells generally showed peak responses at the same time as human peripheral blood leukocytes after stimulation in vitro. Blastogenic responses with purified antigen were comparable to those with the whole-cell vaccine or sonic extract. Such antigens from Legionella provide a useful material for inducing responses in vitro as a correlate of cellular immunity to these bacteria.     

Detection of antibodies to Legionella pneumophila in immune guinea pig serum by solid-phase immunofluorescence.

A semiautomated solid-phase immunofluorescence apparatus (FIAX; International Diagnostic Technology, Santa Clara, Calif.) was utilized to develop a rapid method for detection of antibody to Legionella pneumophila. The sera from guinea pigs immunized with a mixture of killed L. pneumophila and Freund complete adjuvant displayed markedly enhanced antibody activity as measured by FIAX when compared with that obtained from adjuvant-injected or unimmunized animals. A correlation was observed between FIAX net fluorescence units and microagglutination titers of serum samples obtained from immunized animals. Within-run and between-run coefficients of variation performed on selected immune serum samples were low. These results demonstrated that the FIAX method could readily and reproducibly detect Legionella-specific antibodies in the sera of actively immunized animals and suggest the possibility of a broader application of FIAX in the serological detection of exposure to L. pneumophila antigen.     

The course of Legionella pneumonia in guinea pigs after inhalation of various quantities of L. pneumophila

The course of legionella pneumonia in guinea pigs after infection with various quantities of virulent L. pneumophila serogroup 1 organisms by aerosol exposure was investigated. The clinical course, histopathological characteristics, manifestations in the lungs and clearance of the legionella organisms from the lungs and spleen were followed. Four groups were exposed to 4.3 X 10(4), 4.7 X 10(5), 5.0 X 10(6) and 1.0 X 10(8) aerosolized legionellae, respectively. The most striking clinical symptoms were fever and weight loss, which were found in 67-94% and 33-100% of the animals, depending on the dose of L. pneumophila organisms administered. Spontaneous death occurred only in animals receiving the highest dose and always within 10 days. All animals exhibited exponential growth of legionella organisms in the lungs. Maximal growth occurred 5 to 7 days after exposure and varied from 9.3 X 10(7) to 7.4 X 10(8) organisms/both lungs. Twenty-two days after exposure, legionellae could still be cultured from lung tissue. Between 2 and 7 days after exposure, the spleen cultures were positive for legionellae in 41% of the animals receiving the lowest dose and in 83% of all other animals; legionellae could no longer be cultured from spleen tissue after day 7. Depending on the dose, peripherally localized areas of bronchopneumonia increased in size with time, tending to become confluent lobar pneumonia. The microscopic changes were not related to the number of inhaled organisms. In the cellular infiltrate, PMN predominated until day 7 and macrophages thereafter. Seroconversion was found in all animals that survived greater than 7 days. The present animal model closely mimics the course of events in human legionella pneumonia, thus enabling us to further study the factors involved in host resistance against legionella as well as the efficacy of various antimicrobial agents in normal and immunosuppressed animals.     

Immunostimulation by Legionella pneumophila antigen preparations in vivo and in vitro.

Injection of Legionella pneumophila antigen, either killed vaccine or soluble sonicate thereof, resulted in an enhanced antibody response by mouse spleen cells to sheep erythrocytes as determined by the hemolytic plaque assay. Enhancement was dose dependent and reached a peak response at a concentration of 10(7) bacteria or 50 micrograms of sonicate per animal. Larger doses of antigen were less stimulatory or even depressed the antibody response. Similar enhancement of antibody formation by normal spleen cell cultures to sheep erythrocytes in vitro occurred in the presence of graded amounts of L. pneumophila vaccine or sonicate. In addition, the L. pneumophila antigen stimulated enhanced background antibody formation in vitro in the absence of sheep erythrocytes or specific antigen. It appeared likely that the immunoenhancing activity of the L. pneumophila extract may be unrelated to the presence of lipopolysaccharide since boiling the antigen preparation eliminated much of the antibody-enhancing properties of the extract. A large-molecular-weight surface component from L. pneumophila was also immunomodulatory in vitro. Immunostimulation appeared to be related to effects on macrophages since adherent spleen cell populations rich in macrophages, when derived from spleen cell suspensions incubated with L. pneumophila antigen in vitro, stimulated enhanced antibody formation by normal mouse spleen cells in coculture experiments. Further investigations concerning the mechanism of immunomodulation by L. pneumophila antigen in vivo and in vitro appear to be warranted.     

Cellular and humoral immunity in guinea pigs to two major polypeptides derived from hepatitis B surface antigen

Guinea pigs immunized with hepatitis B surface antigen (HBsAg), types adw, adr and ayw, and with two major polypeptides derived from HBsAg/adw developed cell-mediated immunity as determined by the macrophage migration inhibition assay. Peritoneal exudate cells from animals immunized with a 22000- or a 25000-mol. wt. polypeptide derived from HBsAg/adw showed significant migration inhibition after challenge with either polypeptide or with purified HBsAg. Significant inhibition of macrophage migration was not observed when polypeptide-sensitized cells were challenged with normal human serum or with normal human liver extract. Similarly, a cell-mediated immune response was not observed in peritoneal exudate cells from animals sensitized to normal human serum or normal human liver extract which were challenged with either of the polypeptides. The humoral immune response to either of the polypeptides, as measured by radioimmunoassay, was substantially lower than that observed in animals immunized with intact particles. This apparent difference between cellular and humoral responses suggests that the macrophage migration assay is a sensitive indicator of the immunogenicity of the smaller mol. wt. HBsAg-derived polypeptides in guinea pigs.     

NOD1 and NOD2 regulation of pulmonary innate immunity to Legionella pneumophila

The role of nucleotide‐binding oligomerization domain‐1 (NOD1) and nucleotide‐binding oligomerization domain‐2 (NOD2), cytoplasmic receptors which detect bacterial cell wall molecules, in pulmonary innate immune responses is poorly understood. We determined that both NOD1 and NOD2 detect heat‐killed Legionella and stimulate NF‐κb and IFN‐β promoter activity using an in vitro luciferase reporter system. We next infected NOD1‐ and NOD2‐deficient animals with aerosolized Legionella pneumophila. At 3 days post infection, Nod1^?/? mice had impaired bacterial clearance compared to WT controls. In addition, at 4 h and 24 h, Nod1^?/? mice had impaired neutrophil recruitment to the alveolar space. In contrast, increased lung neutrophils were seen in the Nod2^?/? animals at 24 h. Analysis of cytokine production at 4 h post infection revealed a significant decrease in proinflammatory cytokines in the Nod1^?/? animals when compared to WT animals. In contrast, increased 4‐h proinflammatory cytokines were seen in the Nod2^?/? animals. Furthermore, the lungs of both Nod1^?/? and Nod2^?/? mice had significantly increased pro‐inflammatory cytokine levels at 24 h, suggesting possible suppressive roles for later stages of infection. Together, our data suggest that although both NOD1 and NOD2 can detect Legionella, these receptors modulate the in vivo pulmonary immune response differently.     

Direct macrophage migration inhibition test in DNCB contact sensitized guinea pigs.

Using dinitrochlorbenzene contact-sensitized guinea pigs, several DNP conjugates have been assayed in the direct macrophage migration inhibition test (MMIT). Although no significant differences could be observed between the carriers used, conjugates prepared from serum proteins and epidermal extracts tended to give the strongest inhibition of macrophage migration. Conjugates prepared from cells cultured in vitro in the presence of hapten did not cause more inhibition than control conjugates prepared in the absence of cell metabolism. The direct MMIT showed statistical differences between sensitized and nonsensitized groups of guinea pigs. However, none of the conjugates permitted conclusions to be drawn with regard to individual animals.     

Macrophage-activating T-cell factor(s) produced in an early phase of Legionella pneumophila infection in guinea pigs.

Protective immunity of guinea pigs against Legionella pneumophila was studied by infecting the animals with a sublethal dose (about 2 x 10(4) CFU) of the organism. The bacteria multiplied in the liver, spleen, and lungs up to day 4 after the intraperitoneal infection. The live bacteria in these organs decreased quickly thereafter and were eliminated by day 7. A delayed-type skin reaction and lymphoproliferation of spleen cells to Formalin-killed L. pneumophila were detected from days 5 and 6, respectively, after infection. Peritoneal macrophages obtained from guinea pigs infected 6 days previously inhibited the intracellular growth of L. pneumophila. Antigen-stimulated spleen cell factor prepared from infected guinea pigs inhibited the intracellular growth of the organism in macrophages obtained from uninfected animals. Antigen-stimulated spleen cell factor prepared from spleen cells treated with anti-guinea pig T-cell monoclonal antibody did not inhibit growth. The activity of antigen-stimulated spleen cell factor was labile to pH 2 treatment, and the factor could not be absorbed by L. pneumophila antigen, suggesting that it contains gamma interferon. Our data show that T-cell-mediated immunity begins to work from an early period of infection with L. pneumophila in guinea pigs.     

Serum antibodies to Legionella pneumophila by indirect immunofluorescent antibody test in 269 hemodialysis patients and 353 healthy subjects

Serum antibodies to Legionella pneumophila serogroup I-VI were determined by indirect immunofluorescent antibody test in 269 hemodialysis patients and compared to 353 so called healthy subjects. Antigen suspension was prepared by incubation on B-CYE agar and adding formalin solution, then fixed on a micro slide glass, and reacted with serial serum dilutions. FITC-labeled goat antihuman immunoglobulin was added as a second antibody. 273 (77.3%) cases of control group had a titer of less than 1:4, 6 (1.7%) cases had a titer of 1: 32, and none had a titer of greater than or equal to 1: 64. There was no significant difference in sex and age. 173 (64.3%) hemodialysis patients had a titer of less than 1: 4, 13 (4.9%) cases had a titer of greater than or equal to 1: 64, and 5 (1.9%) cases had a titer of 1: 128, 42 (43.8%) of 96 cases who showed a titer of greater than or equal to 1: 4 had antibodies to Legionella pneumophila serogroup I. The titer in hemodialysis patients were higher than control group (p less than 0.005). The results of this study suspected that acute feverish disease and pneumonia of compromised host such as hemodialysis patients should be always thought of Legionnaires' infection.     

Cell-mediated immunity to bacterial flagellin as assessed by leucocyte adherence inhibition

Leucocyte adherence inhibition (LAI) was used to detect cell-mediated immunity of mice to Salmonella adelaide polymeric flagellin and its monomeric derivative. In the direct LAI technique, antigen inhibited the in vitro adherence to glass of peritoneal cells (PC) from antigen-primed mice which were capable of exhibiting in vivo delayed hypersensitivity reactions to the same antigen. In the indirect technique, primed PC exposed to antigen in vitro released a soluble factor, which inhibited the adherence of normal PC. Production of the factor was prevented by prior treatment of primed PC with anti-theta serum, indicating the participation of T-lymphocytes. The LAI reaction could be blocked by serum from mice which had been re-injected with antigen 72 h after a priming injection. Features of the production and biological properties of serum blocking activity suggest that it may be attributed to antigen-antibody complexes.     

Zinc-dependent cytoadherence of Legionella pneumophila to human alveolar epithelial cells in vitro

Microbial adherence to host cells is an early key step in the establishment of infection. During the course of Legionnaire's disease, Legionella interactions with host cells are best documented for resident macrophages. However, L. pneumophila can also replicate within type I and type II pneumocytes, which cover almost the entire alveolar surface. In the presence of zinc, we observed a significant and concentration-dependent increase in L. pneumophila adherence to and invasion of type II pneumocytes. The zinc-dependent adherence mechanism seemed to be host-cell-independent, as a similar increase in cytoadherence was observed with macrophages. We also found that zinc-dependent adherence of L. pneumophila appears to involve recognition of zinc-binding pneumocyte receptors by a bacterial adhesin, and heparan-sulfated host cell receptors, but not type IV pili.     

Zinc-dependent cytoadherence of Legionella pneumophila to human alveolar epithelial cells in vitro

Microbial adherence to host cells is an early key step in the establishment of infection. During the course of Legionnaire's disease, Legionella interactions with host cells are best documented for resident macrophages. However, L. pneumophila can also replicate within type I and type II pneumocytes, which cover almost the entire alveolar surface. In the presence of zinc, we observed a significant and concentration-dependent increase in L. pneumophila adherence to and invasion of type II pneumocytes. The zinc-dependent adherence mechanism seemed to be host-cell-independent, as a similar increase in cytoadherence was observed with macrophages. We also found that zinc-dependent adherence of L. pneumophila appears to involve recognition of zinc-binding pneumocyte receptors by a bacterial adhesin, and heparan-sulfated host cell receptors, but not type IV pili.     

Secondary immunity to Legionella pneumophila and Th1 activity are suppressed by delta-9-tetrahydrocannabinol injection.

Resistance to infection with Legionella pneumophila is primarily dependent upon cell-mediated immunity rather than humoral immunity. Recent evidence suggests that activation of cell-mediated immunity depends on Th1 cells and activation of humoral immunity depends on Th2 cells. In this report, delta 9-tetrahydrocannabinol (THC), the major psychoactive cannabinoid of marijuana and an immunomodulator, suppressed development of secondary immunity to L. pneumophila, which correlated with a reduction in Th1 activity. BALB/c mice, infected with a primary sublethal dose of L. pneumophila, developed resistance to a larger challenge infection 3 to 4 weeks later. However, intravenous injection of THC (4 mg/kg of body weight) 1 day prior to primary infection resulted in increased mortality after the challenge infection. The level of anti-L. pneumophila antibodies in serum increased in both THC-treated and control mice; however, in the THC group IgG1 antibodies which are stimulated by Th2 cells were elevated while Th1-regulated, IgG2a antibodies were depressed. Furthermore, cultured splenocytes from THC-treated mice had less L. pneumophila-specific lymphoproliferation, indicating a deficiency in cell-mediated immunity. Normal mouse splenocytes treated in vitro with THC and pokeweed mitogen showed suppressed production of gamma interferon, a cytokine associated with Th1 cells, but increased production of interleukin 4, a cytokine produced by Th2 cells. Splenocytes from THC-treated mice, stimulated in vitro with either pokeweed mitogen or anti-CD3 antibodies, also produced less gamma interferon, indicating less Th1 activity in these mice. These results suggest that THC decreases the development of anti-L. pneumophila immunity by causing a change in the balance of Th1 and Th2 activities.     

Differential nitric oxide (NO) production by macrophages from mice and guinea pigs infected with virulent and avirulent Legionella pneumophila serogroup 1

l-arginine-dependent reactive nitrogen intermediates have been identified as macrophage cytotoxic effector molecules against intracellular pathogens. To determine its role, ex vivo production of NO by peritoneal macrophages of C3H/HeN mice and Dunkin–Hartley guinea pigs infected intraperitoneally with a virulent and isogenic avirulent Legionella pneumophila serogroup 1 strain was compared with bacterial clearance from the lungs. While the virulent strain was cleared from mice lungs, the guinea pigs died within 96 h. In vivo infection with both strains resulted in the production of NO by mouse peritoneal macrophages ex vivo. In contrast, guinea pig macrophages did not produce detectable NO. In addition, infection by the avirulent strain led to the production of significantly more NO by mouse macrophages than the virulent parent strain, irrespective of stimulation with lipopolysaccharide (LPS) and/or interferon-gamma (IFN-γ). These results suggest that resistance to Leg. pneumophila infection may depend on the production of NO by host macrophages.     

Formed blood elements in peritoneal effusion and the macrophage migration inhibition test in healthy guinea pigs and in guinea pigs with experimental allergic encephalomyelitis.

An attempt was made to correlate the percentages of macrophages, lymphocytes and granulocytes in the peritoneal effusion in healthy guinea pigs and guinea pigs with experimental allergic encephalomyelitis (EAE), with the macrophage migration inhibition (MMI) test. Varying percentages of the cells had no influence on values of MMI. Similarly, in guinea pigs with EAE, percentages of formed elements in peritoneal effusion were not correlated with intensity of MMI or with histopathologic lesions in the brain and spinal cord. It is suggested that the observed differences are due to individual immunologic responsiveness of animals and, probably, to other hitherto unknown mechanisms.