J Med Microbiol 58 (2009), 723-730; DOI: 10.1099/jmm.0.007476-0
© 2009 Society for General Microbiology
ISSN 0022-2615
Experimental Legionella longbeachae infection in intratracheally inoculated mice
Ivana Gobin1,
Milorad Susa1,
Gabrijela Begic1,
Elizabeth L. Hartland2 and
Miljenko Doric1
1 Department of Microbiology and Parasitology, Medical Faculty, University of Rijeka, B. Branchetta 20, 51000 Rijeka, Croatia
2 Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria 3010, Australia
Correspondence
Ivana Gobin
ivanagobin{at}yahoo.co.uk
Received October 22, 2008
Accepted February 12, 2009
This study established an experimental model of replicative Legionella longbeachae infection in A/J mice. The animals were infected by intratracheal inoculation of 103–109 c.f.u. L. longbeachae serogroup 1 (USA clinical isolates D4968, D4969 and D4973). The inocula of 109, 108, 107 and 106 c.f.u. of all tested L. longbeachae serogroup 1 isolates were lethal for A/J mice. Inoculation of 105 c.f.u. L. longbeachae caused death in 90 % of the animals within 5 days, whilst inoculation of 104 c.f.u. caused sporadic death of mice. All animals that received 103 c.f.u. bacteria developed acute lower respiratory disease, but were able to clear Legionella from the lungs within 3 weeks. The kinetics of bacterial growth in the lungs was independent of inoculum size and reached a growth peak about 3 logarithms above the initial inoculum at 72 h after inoculation. The most prominent histological changes in the lungs were observed at 48–72 h after inoculation in the form of a focal, neutrophil-dominant, peribronchiolar infiltration. The inflammatory process did not progress towards the interstitial or alveolar spaces. Immunohistological analyses revealed L. longbeachae serogroup 1 during the early phase of infection near the bronchiolar epithelia and later co-localized with inflammatory cells. BALB/c and C57BL/6 mice strains were also susceptible to infection with all L. longbeachae serogroup 1 strains tested and very similar changes were observed in the lungs of infected animals. These results underline the infection potential of L. longbeachae serogroup 1, which is associated with high morbidity and lethality in mice.
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INTRODUCTION
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Legionella longbeachae was classified as a member of the family Legionellaceae in 1981 (McKinney et al., 1981). There are two serogroups of L. longbeachae and serogroup 1 is most commonly associated with disease in humans (Bibb et al., 1981; Cameron et al., 1991). The pathogen has been identified as a cause of lower respiratory infection in both normal and immunocompromised individuals (O'Connor et al., 2007; Phares et al., 2007). In Europe and the USA, L. longbeachae is rarely implicated in human disease, but the organism appears to be responsible for up to half of the cases of legionellosis in Australia (Lanser et al., 1990; Lim et al., 1989; Steele et al., 1990) and Thailand (Phares et al., 2007). Epidemiological exploration of the source of L. longbeachae serogroup 1 has indicated potting soil as the major reservoir of infection (Cameron et al., 1991; Steele et al., 1990). L. longbeachae serogroup 1 was isolated from commercial potting mixes and the soil of potted plants in Australia, the USA and Japan (CDC, 2000; Hughes & Steele, 1994; Koide et al., 1999). Therefore, it is believed that transmission of L. longbeachae serogroup 1 from the environment to humans may occur via inhalation of aerosols from potting soils containing the organism. To investigate the virulence of different Legionella species, several in vitro and some in vivo experimental models have been developed. L. longbeachae serogroup 2 has been shown to replicate in guinea pig macrophages and A/J, C57BL/6 and DBA/2 mouse peritoneal macrophages (Izu et al., 1999). L. longbeachae serogroup 1 replicates in U937 cells (Doyle & Heuzenroeder, 2002) but is unable to replicate in Mono Mac 6 cells, in Acanthamoeba castellanii (Neumeister et al., 1997) or in Hartmannella vermiformis (Wadowsky et al., 1991). Recently, it was shown that L. longbeachae serogroup 1 has a different intracellular life cycle to Legionella pneumophila, where, unlike L. pneumophila, the L. longbeachae-containing vacuole possesses markers of both early and late endosomes (Asare & Abu Kwaik, 2007). It has also been shown that a L. longbeachae serogroup 1 clinical isolate was capable of causing death and disease in guinea pigs exposed to an aerosol dose of the organism, similar to the observation with L. pneumophila (Cianciotto et al., 1990). Furthermore, Doyle et al. (2001) showed that there are distinct virulence groupings of L. longbeachae serogroup 1 strains based on the severity of disease produced in a guinea pig model. Previous studies have shown that the inbred mouse strains C57BL/6, BALB/c, C3H/HeN and DBA/2 are resistant to L. pneumophila infection, and that only A/J mice are susceptible (Brieland et al., 1994; Izu et al., 1999). In inbred mouse strains, permissiveness to intracellular replication of L. pneumophila is associated with polymorphisms in the Naip5 allele and flagellin-mediated early activation of caspase 1 in mouse macrophages (Molofsky et al., 2006; Wright et al., 2003). In contrast to L. pneumophila, different mouse strains, including A/J, BALB/c and C57Bl/6, are susceptible to L. longbeachae infection, and therefore the genetic susceptibility of mice to infection by L. longbeachae is apparently independent of the allelic polymorphism of Naip5 (Asare et al., 2007). In this study, we examined pathological changes in the murine lung caused by experimental L. longbeachae infection to improve our knowledge of the pathological mechanisms implicated in infection. Through this work, we have established a reliable murine model of legionellosis caused by L. longbeachae serogroup 1 to aid further investigations into the pathogenesis of L. longbeachae infections.
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METHODS
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Animal care.
Female and male specific-pathogen-free, 6–10-week-old A/J, BALB/c and C57BL/6 mice were used in the experiments. The animals were obtained from the Central Animal Facility of the Medical Faculty (University of Rijeka, Croatia), kept in plastic cages and given standard laboratory rodent food and water ad libitum. The experiments were conducted in accordance with the guidelines found in the International Guiding Principles for Biomedical Research Involving Animals. The Ethical Committee at the University of Rijeka approved all of the animal experiments described here.
Bacterial inoculum.
The three strains of L. longbeachae serogroup 1 (D4968, D4969 and D4973) were clinical isolates from California, Oregon and Washington State, USA, and were kindly provided by Dr Yousef Abu Kwaik (Department of Microbiology and Immunology, University of Louisville, USA; CDC, 2000). Bacteria were stored at –80 °C in 10 % glycerol broth. Legionella cells were used directly from buffered charcoal yeast extract agar plates (BCYE-
; Merck). A stock solution containing approximately 2x1010 c.f.u. Legionella ml–1 was used for survival experiments and 108 c.f.u. ml–1 for other experiments, and was used immediately. The number of viable bacteria in the inoculum was confirmed by enumeration of the bacteria on BCYE- agar.
Animal inoculation.
Mice were inoculated intratracheally according to a previously described protocol (Brieland et al., 1994; Susa et al., 1998). Briefly, the mice were anaesthetized by intraperitoneal injection of ketamine (2.5 mg per mouse) and the trachea was isolated. A 50 µl water suspension containing a total of 103–109 c.f.u. was inoculated directly into the trachea. Control animals were mock-infected with heat-inactivated bacteria or inoculated with sterile water.
Assessment of virulence and quantification of L. longbeachae in mice.
For enumeration of bacterial load at the indicated time points following inoculation, the mice (three animals per group) were euthanized by cervical dislocation. Independent experiments were performed at least three times. To enumerate viable bacteria in the lungs of infected mice, the organs were removed aseptically, minced and homogenized with 5 ml sterile water, centrifuged and resuspended in 1 ml sterile water. Determination of c.f.u. in the different tissues was carried out by using a serial dilution method. Results were expressed as the number of c.f.u. per lung homogenate.
Histopathological analysis and immunostaining.
Following euthanasia, the lungs were instilled with buffered 4 % paraformaldehyde for 10 min and placed into 4 % paraformaldehyde for fixation. Fixed lungs were mounted in paraffin, and sections were cut and prepared for staining. Sections from the lungs were stained with haematoxylin and eosin. Alternatively, the hydrated lung sections were treated with 3 % hydrogen peroxide in 70 % methanol to block endogenous peroxidase. Slides were then incubated with an appropriate dilution of primary antibody (rabbit anti-L. longbeachae polyclonal serum, kindly provided by Dr Yousef Abu Kwaik). Controls were performed in which non-specific rabbit serum was substituted for the primary antibody to ensure the specificity of the staining. The slides were incubated with a biotin-labelled secondary anti-rabbit antibody (Vectastain Elite ABC kit; Vector Laboratories) for an additional 60 min, washed and incubated with streptavidin–peroxidase for 20 min. Visualization was achieved by incubation with 0.05 % 3.3'-diaminobenzidine tetrahydrochloride. The slides were counterstained with Mayer's haematoxylin.
Evaluation of histology and immunohistochemistry.
The sections were analysed using an Olympus BX51 microscope. Histology and immunohistology were analysed at magnifications of x100, x400 and x1000. The figures show representative examples from five to seven experiments using three mice per group.
Statistical analysis of data.
The number of bacteria in each experiment was estimated by dividing the sum of bacterial counts for different dilutions by the sum of the dilution factors. Datasets were analysed using the Kruskal–Wallis comparison test followed by Student's t-test. Significance was assigned where P <0.05.
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RESULTS AND DISCUSSION
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Assessment of virulence of L. longbeachae serogroup 1 isolates
Experimental animals (five to ten animals per group) were challenged with 103–109 c.f.u. L. longbeachae (Fig. 1
). Irrespective of the L. longbeachae isolate (D4968, D4969 or D4973) used for inoculation, the inocula of 109, 108, 107 and 106 c.f.u. Legionella were lethal for all tested mice. In groups of mice challenged with 105 c.f.u. of each L. longbeachae strain, approximately 10 % of the animals eventually cleared the infection and survived. An inoculum of 105 c.f.u. L. longbeachae therefore appeared to be at the limit of control by the host immune system. An inoculum of 104 c.f.u. had a moderate effect, although sporadic death of mice was still observed in each test group. Mice infected with 103 c.f.u. of each L. longbeachae strain developed a respiratory infection but all survived. In contrast to the results of Doyle et al. (2001), we did not observe differences in virulence between the tested strains. However, in vivo, L. longbeachae showed at least a 100-fold higher infectivity potential than L. pneumophila for A/J mice (Susa et al., 1998). Such a low inoculum leading to a lethal infection is, to our knowledge, unusual for Legionella species. Interestingly, whilst most other Legionella infections are associated with a contaminated water source, L. longbeachae resides in soil, and our data suggest that the aspiration of even very low doses of L. longbeachae from soil may lead to infection in humans.
Replication of L. longbeachae serogroup 1 in the lungs of mice
In order to determine the in vivo growth kinetics of different L. longbeachae serogroup 1 isolates in the lungs of experimental mice (A/J, BALB/c and C57BL/6), the animals were inoculated with 103 or 105 c.f.u. bacteria and the number of c.f.u. in the lungs was determined at different time points (three mice per group) (Fig. 2). Within the lungs, all tested strains of L. longbeachae showed exponential growth during the first 3 days post-infection (p.i.) (Fig. 2a, b). Mice infected with 103 c.f.u. were able to reduce the bacterial burden in the lungs below the detection level of the assay (Fig. 2a, c). However, in some animals infected with 103 c.f.u., we were able to detect L. longbeachae in the lungs for up to 21 days after intratracheal inoculation (data not shown). Again, there were no significant differences between the tested strains of L. longbeachae serogroup 1 (Fig. 2c, d). In addition, irrespective of the initial inoculum size or genetic background of the mouse strain, the in vivo growth index of L. longbeachae was approximately 1000, which was measured by comparison of bacterial yield in the lung 72 and 2 h after inoculation. As already demonstrated, L. longbeachae serogroup 1 replicates equally well in the lungs of A/J, BALB/c and C57BL/6 strains of mice (Asare et al., 2007). In summary, we demonstrated that all of the tested L. longbeachae strains caused a productive pulmonary infection after inoculation of only 103 c.f.u.
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Fig. 2. (a, b) In vivo growth of different L. longbeachae isolates (D4968, D4969 and D4973) in the lungs of A/J mice inoculated with 103 c.f.u. (a) and 105 c.f.u. (b). (c, d) In vivo growth of L. longbeachae D4968 in the lungs of A/J, BALB/c and C57BL/6 mice inoculated with 103 c.f.u. (c) and 105 c.f.u. (d). The graphs show representative c.f.u. data obtained from three lungs of three to five experiments and are presented as the median value and the range of maximal and minimal values.
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Histopathological changes in the lungs during infection with L. longbeachae serogroup 1
In this study, we characterized lung lesions caused by L. longbeachae serogroup 1. All strains tested caused very similar changes in the lungs of A/J, BALB/c and C57BL/6 mice (data not shown). Fig. 3 shows the lung histology of A/J mice after intratracheal inoculation with 105 c.f.u. viable or heat-inactivated L. longbeachae serogroup 1 D4968. Two hours after intratracheal inoculation, the first changes were seen in the columnar epithelium of the bronchioles. The epithelial cells were rounded on the apical side, reduced in size and had partially lost their pseudostratified morphology. The muscularis mucosa of the basement membranes of the bronchioles was rarefied and enlarged. No infiltration was seen in the muscularis mucosa or in the peribronchiolar regions or luminal spaces of the bronchioles. At 72 h after intratracheal inoculation of L. longbeachae serogroup 1, the columnar epithelium was destratified and reduced to columnar basal epithelial cells. The smooth muscle layers of the muscularis mucosa were thickened, infiltrated with polymorphonuclear and mononuclear cells, and showed necrotic degeneration. The peribronchiolar and interstitial spaces of the alveoli close to the bronchioles were predominantly infiltrated with neutrophils, but there was no progression of infiltration towards the alveolar sacs or to the centrifugal interstitial spaces. The inflammatory cells were recruited only around the bronchioles, giving an impression of focal infiltration. The luminal sites of the bronchioles were also infiltrated with polymorphonuclear leukocytes. Blood vessel walls and intimas were thickened and infiltrated with inflammatory cells. As the lethality of mice receiving 105 c.f.u. L. longbeachae serogroup 1 (strain D4968) was very high, it was not possible to observe the late pathological changes in their lungs. Therefore, we also followed the histopathological changes in the lungs of mice inoculated with 103 c.f.u. L. longbeachae serogroup 1 (strain D4968) (Fig. 4). Although the changes were delayed, they were very similar to those of animals receiving an inoculum of 105 c.f.u., i.e. initial morphological changes in the epithelial cells between 24 and 48 h p.i., and focal peribronchiolar inflammatory cell infiltration with basement membrane injury 3–7 days p.i. (Fig. 4). Restoration of lung parenchyma was observed not earlier than 14–21 days after infection and correlated with the ability of mice to control the in vivo growth of L. longbeachae serogroup 1 in the lungs. To our knowledge, this is the first report that L. longbeachae serogroup 1 causes bronchopneumonia in different mouse strains. Recently, histopathological examination of the lungs of patients who had died of L. longbeachae infection indicated a purulent bronchopneumonia (Kümpers et al., 2008). This is in contrast to L. pneumophila pneumonia in A/J mice. Brieland et al. (1994) showed that L. pneumophila caused changes mainly in the alveoli, and that the bronchi and bronchioles were essentially normal.
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Fig. 3. Representative histopathology of the lungs during infection of A/J mice with 105 c.f.u. viable or heat-inactivated (mock infection) L. longbeachae serogroup 1 (strain D4968). Experiments were repeated three times. The sections were analysed under x100 (a) or x400 (b) magnification. The lungs of mock-inoculated mice did not show any histopathological changes at the analysed time points. al, alveolar spaces; bm, basement membrane; br, bronchioli; ep, bronchiolar columnar epithelium; gc, goblet cell; ic, inflammatory cells.
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Fig. 4. Representative analysis of lung histopathology during an infection of A/J mice inoculated with 103 c.f.u. viable L. longbeachae. The sections were stained with haematoxylin and eosin. At the indicated time points, the histology was analysed under x100 magnification.
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Immunohistochemistry for detection of L. longbeachae serogroup 1 (strain D4968) in lung parenchyma
The pathological changes in lung parenchyma after intratracheal inoculation of L. longbeachae serogroup 1 suggested that the changes correlated with the localization of bacteria. Therefore, we performed immunohistochemistry of the lungs to determine the exact localization of L. longbeachae. Two hours after intratracheal inoculation of 105 c.f.u. L. longbeachae (strain D4968), single stained cells, most probably macrophages engulfing the bacteria, were detected in close proximity to the respiratory epithelium. With progression of infection, the positively stained cells in the lung parenchyma increased in number. Unfortunately, we were not able to observe whether L. longbeachae serogroup 1 also resided within alveolar epithelial cells. In the late stages of infection, L. longbeachae was detected within the focal infiltrates of the bronchioli or inside their lumina (Fig. 5). However, we were not able to detect any infiltrating cells with L. longbeachae either within the basement membranes of the bronchioles or outside the focal peribronchiolar infiltrates. The acute injury of lower respiratory epithelium 2 h after inoculation of bacteria and early co-localization of L. longbeachae with the respiratory epithelium surface structures may indicate a host–pathogen interaction responsible for the morphological rearrangement.
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Fig. 5. Immunohistology of the lungs from A/J mice 2–72 h after intratracheal inoculation of 105 c.f.u. L. longbeachae. Representative lung slices were stained with rabbit polyclonal anti-L. longbeachae serum, and L. longbeachae was visualized using diaminobenzidine (brown) and counterstained with Mayer's haematoxylin. Magnification: (a) x400; (b) x1000. At the higher magnification, intracellular stained bacteria can be visualized. Positively stained cells are indicated by an asterisk. The lungs of mock-inoculated mice did not react with serum at any time point analysed (not shown).
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In conclusion, L. longbeachae serogroup 1 is a highly virulent pathogen in mice. The bronchopneumonia caused by L. longbeachae serogroup 1 was not restricted to a particular genetic background of the host mice. Within 3 days, L. longbeachae serogroup 1 can cause a severe bronchopneumonia characterized by a focal, predominantly neutrophil infiltration of peribronchiolar spaces and bronchiolar lumina, with degeneration of bronchiolar epithelia and basement membranes. Although mice are not natural hosts of L. longbeachae, our data showed that mice are a useful surrogate model for human infection. A drawback of studying host responses to L. pneumophila infection has been the restriction of this species to the A/J mouse background. In contrast, it will be possible to study the pathogenesis of disease caused by L. longbeachae serogroup 1 strains and the importance of particular immune responses to L. longbeachae infection in several mouse backgrounds, in particular C57BL/6 mice, for which there are many knockout and transgenic lines available. Although the ability of L. longbeachae serogroup 1 to infect a range of mouse strains may reflect additional or different virulence factors to L. pneumophila, the fact that serogroup 1 strains of L. longbeachae are largely non-motile may also contribute to the increased virulence of this species over L. pneumophila. It is well documented that mutants of L. pneumophila that lack flagellin have increased virulence in A/J mice and can also infect C57BL/6 mice, similar to L. longbeachae serogroup 1 (Molofsky et al., 2006). In summary, our study characterized an intratracheal mouse infection model for L. longbeachae. This information will be of great value for studying the virulence and pathogenesis of L. longbeachae infections in comparison with L. pneumophila.
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ACKNOWLEDGEMENTS
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This study was financially supported by the Ministry of Science, Education and Sport of the Republic of Croatia (Project 062-0621273-1275). We thank Tibor Andreanszky, University of Rijeka, for his technical assistance. I. G. was supported by an Australian Endeavour Post-doctoral fellowship.
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References
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N. A. Kozak, M. Buss, C. E. Lucas, M. Frace, D. Govil, T. Travis, M. Olsen-Rasmussen, R. F. Benson, and B. S. Fields
Virulence Factors Encoded by Legionella longbeachae Identified on the Basis of the Genome Sequence Analysis of Clinical Isolate D-4968
J. Bacteriol.,
February 15, 2010;
192(4):
1030 - 1044.
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INTRODUCTION
METHODS
), 104 (
), 105 (
), 106 (
), 107 (
), 108 (
) or 109 (•) c.f.u. of L. longbeachae (isolate D4968). The death of animals is plotted as percentage survival rate against the number of days after exposure to bacteria. The graph represents a typical result obtained from five to ten animals per group. The experiments were repeated at least three times for each isolate of L. longbeachae (D4968, D4969 and D4973). Statistical groupings were assigned based on time to death.