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This chapter talks about the role of the flagellum in the observed protection from legionella infection that was investigated by the authors. First, the minimal lethal dose (MLD) of two different strains of Legionella pneumophila was determined by infecting intraperitoneally (i.p.) female A/J mice, aged 4 to 6 weeks, with different doses of an L. pneumophila serogroup 6 strain of human origin (VIR+), or from the L. pneumophila serogroup 1 Corby strain. A flagellar preparation was performed from a 6-day culture of the flagellated VIR+ strain on buffered charcoal-yeast extract (BCYE; Oxoid, Italy) agar plates at 36№C, in humidified atmosphere and 2.5% CO2. Mice were observed for signs of illness and for survival for 15 days. In flagella-immunized mice, no mortality was observed, compared with 100% mortality of similarly challenged mice immunized with the sham-flagellar preparation or unimmunized control mice. Involvement of humoral immune responses in the protective effect due to flagellar immunization was investigated, also considering that flagella are present in most Legionella species and serogroups, and their composition is common in all of them.

Legionella pneumophila causes a severe atypical pneumonia in humans, the so-called Legionnaires' disease. Although the disease mostly occurs in immunocompromised subjects, several virulence factors of Legionella have been described that confer to the microorganism an intrinsic aggressive potential. The ability of this pathogen to replicate in host macrophages is considered a major virulence trait. For this reason, the susceptibility to the experimental infection with L. pneumophila observed in guinea pigs and in selected mouse strains has been generally explained by the permissiveness of macrophages of these animals for Legionella invasion and intracellular replication in vitro, as reported, for elicited peritoneal macrophages from susceptible A/J mice. Researchers found that the i.p. inoculation of virulent Legionella cells from the VIR+ strain was rapidly lethal for A/J mice, since 100 and 60% of the animals acutely died within 2 days following the challenge with 2 X 108 or 2 X 107 bacterial cells. Unchecked TNF-α production upon i.v. Legionella inoculation has recently been reported as the causative factor in the death of Legionella replication-unpermissive BALB/c mice. Accordingly, the protection afforded by IL-10 treatment or TNF-α neutralization in the mice model also indicates that the release of TNF-α, a well-known toxic shock-related cytokine, plays a major role in mediating the lethal effects of the virulent legionellae. Therefore induction of proinflammatory cytokines, in particular TNF-α, besides being an important factor facilitating the resolution of L. pneumophila infections, might also be regarded as a possible contributory factor to the pathogenic action of virulent L. pneumophila strains.

Objectives To compare genotypic methods for epidemiologic typing of Legionella pneumophila serogroup (sg) 1, in order to determine the best available method within Europe for implementation and standardization by members of the European Working Group on Legionella Infections. Methods Coded isolates (114) of L. pneumophila sg 1 comprising one epidemiologically ‘unrelated' (79) and one ‘related' panel of isolates (35) were sent to 12 laboratories in 11 European countries. Analysis was undertaken in each laboratory using one or more of the following methods: ribotyping, restriction fragment length polymorphism analysis, restriction endonuclease analysis, pulsed-field gel electrophoresis (PFGE), PCR using arbitrary/repeat sequence primers (AP-, AP/rep-PCR), and amplified fragment length polymorphism (AFLP) analysis. Results were analyzed visually or using gel analysis software. Each method was assessed for its: index of discrimination ( D ), epidemiologic concordance ( E ), speed of application and ease of use. In addition, phenotypic analysis was performed in two laboratories using monoclonal antibodies (mAbs). Results The D of each of the genotypic methods ranged from 0.840 for ribotyping to 0.990 for PFGE using Sfi I : E ranged from 0.06 for AP- and AP/rep-PCR to 1.00 for ribotyping using Pst I /Eco RI and AFLP: in general, E was inversely related to D. Although offering only limited discrimination ( D =0.838), mAb typing was both rapid and highly epidemiologically concordant ( E =1.00). Conclusions Two methods, PFGE using Sfi l and AFLP, were selected for further study. AFLP is rapid and highly epidemiologically concordant ( E =1.00), but is not highly discriminatory. This method will be developed as a rapid screening tool. PFGE using Sfi l is highly discriminatory but, in the present study, yielded low values of E (0.12–0.71). Attempts will be made to rigorously standardize this method for use as the reference method. Primary screening of isolates by mAb subgrouping is recommended.

Nine unrelated Legionella micdadei strains isolated from clinical and environmental samples have been characterized biochemically, serologically using polyclonal and monoclonal antibodies and by macrorestriction analyses using pulsed-field gel electrophoresis. All strains were positive in the Bromocresol purple spot test and grew as blue colonies on dye-containing media. They were positive for catalase, weakly positive for oxidase, and negative for sodium-hippurate hydrolysis, β-lactamase and gelatinase. None of the strains showed autofluorescence under long-wave ultraviolet light. A panel of six monoclonal antibodies raised against the ATCC strain TATLOCK revealed no significant differences in the surface antigen composition of the L. micdadei strains. None of these monoclonal antibodies reacted with L. maceachernii and L. longbeachae serogroup 2, the only species that cross-react with polyclonal antisera. Each of the nine L. micdadei strains showed individual restriction patterns of the genomic DNA when using both SfiI and NotI restriction enzymes in the pulsed-field gel electrophoresis. Macrorestriction analysis is a valuable tool for studies on the molecular epidemiology of L. micdadei.

Legionella pneumophila is a pathogen that causes severe pneumonia in humans; L. pneumophila serogroup 1 accounts for at least 90% of infections. This is not linked to an environmental predominance of Legionella pneumophila 1, but may be due to a greater virulence of the strain. L. pneumophila sg 5 has also been reported, albeit less frequently, to be a cause of the disease. We report a case of L. pneumophila sg 5 occurring in a large hospital in southern Italy (Apulia region), where both L. pneumophila sg 1 and sg 5 were detected in the water supply; the nosocomial origin was demonstrated by molecular subtyping (PFGE). An environmental investigation, performed immediately after diagnosis of the case of legionellosis, identified a ow L. pneumophila sg 5 contamination level. Our experience highlights that in hospital, risk assessment, in rder to institute control measures for Legionella, should be carried out not only in response to a case of the disease and/or in risk wards only, as described in the Italian Guidelines, but periodically in every ward. The present study confirms that, although in the community L. pneumophila sg 1 is the most frequent strain isolated in both outbreaks and isolated cases, in hospital other serogroups and species may often cause infection because of the high susceptibility of the hosts.

The activity of six intracellular antibiotics, doxycycline, erythromycin, clarithromycin, azithromycin, rifampicin and ciprofloxacin, was tested against 60 strains of Legionella pneumophila (21 of human and 39 of environmental origin). MIC50, MIC90, and MBC values were determined by a microdilution method. Inhibitory and bactericidal activity against human and environmental isolates were similar except for rifampicin, which was 100-fold less active for human strains than for environmental strains, particularly in terms of bactericidal activity. Nevertheless, in general, rifampicin was found to be the most active drug. Among the macrolides tested, clarithromycin showed the greatest activity in MIC assays and erythromycin was the least bactericidal. Azithromycin showed higher MICs and MBCs than the two macrolides, and doxycycline was the least active. The most important factors influencing in-vivo activity of antibiotics are discussed. Even if the in-vitro results cannot be fully extrapolated to activity in vivo, these results indicate the susceptibility of L. pneumophila strains in Italy as a basis for treatment of atypical pneumonia that may be due to Legionella spp.

The capacity of a purified preparation of Legionella pneumophila flagella (FLA) to induce protective immune responses was studied in an A/J mouse model. Animals immunized with FLA promptly mounted an anti-FLA antibody response and also developed a strong activation of both innate and adaptive cell-mediated immunity, as shown by an early release of pro-inflammatory cytokines in the peritoneal cavity, and by a positive cutaneous delayed-type hypersensitivity reaction and in vitro splenic lymphocyte proliferation in response to FLA antigens. Mice treated with FLA either i.v. or i.p. also survived (100% rate) a lethal i.p. challenge with L. pneumophila. Protection induced by FLA lasted for at least 30 days after treatment, but less than 60, and was effective against the challenge with different serogroups of L. pneumophila. Resistance conferred by FLA immunization could be partially transferred to naive animals by the adoptive transfer of immune splenocytes but not by passive immunization with anti-FLA iperimmune sera. The capacity to induce protective immunity was specifically attributable to flagellar components, as demonstrated by the lack of protection in mice immunized with a sham flagella preparation from a non-flagellated bacterial strain or with protease-digested FLA. In addition, heat-denatured FLA was inactive, suggesting loss of immunogenicity following denaturation. The present study provides evidence that L. pneumophila flagellum is strongly immunogenic and capable to stimulate, without adjuvants, early natural and acquired, T-cell-mediated immune responses and to induce significant protection against a lethal bacterial challenge in A/J mice. Antigenic characterization of this bacterial organelle and elucidation of mechanisms underlying flagella-induced protection would be of great value in understanding the immunopathogenesis of the disease and in developing possible therapeutic strategies for human legionellosis.

This international collaborative survey identified culture-confirmed legionellosis in 508 patients with sporadic community-acquired legionellosis. Legionella pneumophila constituted 91.5% of the isolates. Serogroup 1 was the predominant serogroup (84.2%), and serogroups 2-13 (7.4%) accounted for the remaining serogroups. The Legionella species most commonly isolated were L. longbeachae (3.9%) and L. bozemanii (2.4%), followed by L. micdadei, L. dumoffii, L. feeleii, L. wadsworthii, and L. anisa (2.2% combined). L. longbeachae constituted 30.4% of the community-acquired Legionella isolates in Australia and New Zealand.

The occurrence of legionnaires' disease has been described previously in passengers of cruise ships, but determination of the source has been rare. A 67-year-old, male cigarette smoker with heart disease contracted legionnaires' disease during a cruise in September 1995 and died 9 days after disembarking. Legionella pneumophila serogroup 1 was isolated from the patient's sputum and the ship's water supply. Samples from the air-conditioning system were negative. L. pneumophila serogroup 1 isolates from the water supply matched the patient's isolate, by both monoclonal antibody subtyping and genomic fingerprinting. None of 116 crew members had significant antibody titers to L. pneumophila serogroup 1. One clinically suspected case of legionnaires' disease and one confirmed case were subsequently diagnosed among passengers cruising on the same ship in November 1995 and October 1996, respectively. This is the first documented evidence of the involvement of a water supply system in the transmission of legionella infection on ships. These cases were identified because of the presence of a unique international system of surveillance and collaboration between public health authorities.

A study has been carried out on the action of cytoskeleton and metabolic inhibitors on intracellular multiplication in HeLa cells of a virulent strain of Legionella pneumophila serogroup 6. The effects of the substances were separately tested on both penetration and intracellular multiplication of L. pneumophila. Only cytochalasin A and 2-deoxy-d-glucose (2dG) affected bacterial internalisation, whereas intracellular multiplication was inhibited by cytochalasins A, B, C, D and J (D being the most active) and by 2dG with a dose-response effect. The action of 2dG was counteracted by 50 mM glucose. Experiments carried out with cytochalasin D and a rhodamine-phalloidin conjugate showed the involvement of cytoskeletal elements in intracellular multiplication of Legionella; compounds acting on microtubules had no effect.

A fatal pneumonia due to Legionella pneumophila was diagnosed in a young calf reared in a dairy herd located in northern Italy. Clinical symptoms consisted of watery diarrhea, hyperthermia, anorexia, and severe dyspnea. The pathological and histological findings were very similar to those observed in human legionellosis. Legionella pneumophila serogroup 1 (SG1) and SG10 were isolated from the calf’s lung, and L. pneumophila SG1 was isolated from the calf’s liver. L. pneumophila SG1 was also demonstrated in the lung tissue by immunofluorescence and immunohistochemical examinations. Nine of 10 L. pneumophila SG1 isolates belonged to the Olda subtype, and 1 belonged to the Camperdown subtype. A very low prevalence of antibodies to Legionella was detected in cows and calves reared in the same herd. Cultures of aqueous sediment of an old electric water heater which supplied hot water for the feeding of the calves yielded L. pneumophila SG1. Four of the colonies tested belonged to the Olda subtype. Ten clinical and four environmental isolates were examined for the presence of plasmids. Nine of them were also examined by pulsed-field gel electrophoresis assay, and the same patterns were found for L. pneumophila SG1 Olda strains isolated from the calf and from the electric heater. This is the first report of a documented case of a naturally occurring Legionella pneumonia in an animal. Cattle probably act as accidental hosts for legionellae, much the same as humans.

Nine unrelated Legionella micdadei strains isolated from clinical and environmental samples have been characterized biochemically, serologically using polyclonal and monoclonal antibodies and by macrorestriction analyses using pulsed-field gel electrophoresis. All strains were positive in the Bromocresol purple spot test and grew as blue colonies on dye-containing media. They were positive for catalase, weakly positive for oxidase, and negative for sodium-hippurate hydrolysis, beta-lactamase and gelatinase. None of the strains showed autofluorescence under long-wave ultraviolet light. A panel of six monoclonal antibodies raised against the ATCC strain TATLOCK revealed no significant differences in the surface antigen composition of the L. micdadei strains. None of these monoclonal antibodies reacted with L. maceachernii and L. longbeachae serogroup 2, the only species that cross-react with polyclonal antisera. Each of the nine L. micdadei strains showed individual restriction patterns of the genomic DNA when using both SfiI and NotI restriction enzymes in the pulsed-field gel electrophoresis. Macrorestriction analysis is a valuable tool for studies on the molecular epidemiology of L. micdadei.

The detection of travel-associated legionellosis can be extremely difficult; hence, an extensive case investigation is recommended in pneumonia-striken travellers and tourists, who are particularly at risk of acquiring the disease. On the Island of Ischia (Isola d'Ischia, Naples, Italy) a total of six cases of Legionnaires' disease occurred from 1986 to 1990. All patients (one man and two women from Germany, one Austrian woman, one Swiss man, and one Italian woman) had taken thermal baths and stayed in local hotels; they all experienced severe pneumonia, and three of them died. These cases were associated with hotels, and the hot-water supply was presumed to have transmitted the infection. Remedial procedures were applied to the hot-water plumbing of the hotels according to the WHO recommendations and were proved to be effective. The occurrences described in this paper stress the importance of rapid and accurate reporting of diagnosed cases to the country where the infection was probably acquired, in order to ensure early detection of endemic foci and emerging clusters of legionellosis.

Summary. The growth inhibiting activity of transferrins, citrate, 2-2′ dipyridyl and desferrioxamine methanesulphonate towards Legionella spp. and their serogroups was investigated. The inhibitory activity of all these compounds depended upon the iron-free state of the molecules and was abolished by saturation with iron. No bactericidal effect by transferrins was observed at concentrations up to four times the minimal bacteriostatic concentration. No interaction of transferrins with the legionella cell surface was detected by direct or indirect fluorescence assay, or by dialysis culture experiments in which transferrin was separated from the bacterial cells. The demonstration of a siderophore-like activity in supernates of iron-deficient legionella cultures may account for the ability of Legionella spp. to multiply in conditions of iron restriction.

After having eaten dinner many customers of a restaurant in Dolo (Venice) developed gastroenteritis with diarrhoea and vomiting. Strains of non O group 1 Vibrio cholerae were isolated from rectal swabs of four customers. Epidemiological investigations revealed an association between the illness and raw oyster consumption (p = 0.008). Also, an association was found between age and illness among males (p = 0.034). As a consequence of this outbreak, a gastroenteritis surveillance programme was run for three months by health authorities in the Regione Veneto. The results indicate that, although non 0-1 V. cholerae does not account for severe pathology, it causes mild gastroenteritis and asymptomatic infections in developed countries.

A microorganism antigenically identified as Legionella micdadei but showing a cellular fatty acid profile distinct from that described previously for this species and more similar to that of L. bozemanii has been studied by phenotypic characterization, crossed immunoelectrophoresis, gas-liquid chromatography, and transmission electron microscopy. Although the phenotypic characters, the crossed immunoelectrophoresis, and the ultrastructural features were similar to those of L. micdadei, the fatty acid composition differed significantly from this species; moreover it differed also from L. bozemanii, even though it was quantitatively more similar.

Acute-phase and convalescent-phase sera of 66 children, aged 3 months to 12 years, with neurologic disorders of unknown etiology were tested against Legioneila pneumophila polyvalent and monovalent antigens (groups 1 to 4). Three significant antibody titer increases were obtained, all in childlren with acute cerebellar ataxia. This neurologic syndrome was characterized by sudden onset of muscle hypotonia and inability to sit or walk, with no other specific neurologic or systemic symptoms. Persisting pharyngitis always preceded ataxia. Fever of short duration was still present. Gastrointestinal disturbance occurred in two of the three children. Abnormal laboratory findings were, not always simultaneously, high ESR and leukocytosis with lymphocytosis. CSF levels and electromyographic findings were normal in two of the children. Two children received oral betamethasone. Recovery was complete within seven to ten days without antibiotic treatment. These studies indicate the possible etiologic role of L pneumophila in acute cerebellar ataxia.

From Oct. 30 to Nov. 7, 1979, 10 people in the Sardinian province of Cagliari had onset of bacteriologically confirmed cholera. Two symptom-free excretors of Vibrio cholerœ O:1 were detected in household contacts of the patients. There were no deaths. All but 1 of the 12 people with V. cholerœ O:1 infection gave a history of recent consumption of marine bivalves known locally as arselle (pelecypods). Triplicate matched neighbourhood controls for each of the first 7 cases identified were also interviewed; none had recently eaten arselle. V. cholerœ O:1 was also recovered from samples of water and bivalves obtained from a lagoon on the outskirts of the city of Cagliari. Arselle had also been implicated as the vehicle of transmission in 1973 in the last outbreak of cholera in Sardinia. It seems unlikely that cholera transmission had persisted locally in the interim.

Outbreaks of febrile illness consistent with legionellosis occurred in successive groups of vacationers at an Adriatic resort in 1980. Illness was associated with one of two hotels used by the groups. A cohort study of guests of the suspect hotel revealed 23 cases of febrile illness with pulmonary symptoms, significant antibody titers to Legionella pneumophila, or both, among 291 persons at risk. Two patients died. Attack rates ranged from 0-19% in the nine cohorts of vacationers and were highest among the most elderly. Febrile illness in the last two cohorts of the season was associated with an antibody titer greater than or equal to 128. L. pneumophila was isolated from shower heads in the suspect hotel, but no association was found between showering and illness or seropositivity. Bacteria identified as L. pneumophila by direct immunofluorescence were also found in water from an adjacent hotel and from the outflow from a common well. No cases were associated with the adjacent hotel.

On the assumption that specific host defences are lower in newborn and infant animals, the susceptibility of CD1 suckling mice to Legionella pneumophila was studied with the hypothesis that this model could detect consistent differences in virulence among Legionella isolates from various clinical and environmental sources. Mice 3-14 days old were indeed markedly susceptible to intraperitoneal challenge with fresh clinical isolates, but not to serially subcultured or type collection strains of L. pneumophila. For example, intraperitoneal inoculation of 10(7) cells of a fresh clinical isolate of L. pneumophila (strain Monza 3) caused 60% mortality of suckling mice in 1 day whereas the same number of cells of a culture-attenuated derivative (strain Monza 3p50) caused10% mortality in15 days. Lethal infection by the 'virulent' Monza 3 strain was strictly dependent on mouse age (no death was observed in mice26 days old), required the inoculation of viable cells and was not related to endotoxin production. The 'virulent' L. pneumophila strain was cleared from mouse lungs less rapidly, while adhering to, and being internalised into the peritoneal exudate cells (PEC) of suckling mice to a greater extent, than the avirulent derivative, as shown by immunofluorescence and confocal microscopy. The Monza 3 strain also induced the production by PEC in vivo of 5-to-10 times more tumour necrosis factor-alpha (TNF-alpha) mRNA than the Monza 3p50 strain. Overall, suckling CD1 mice appear to provide a promising, easily handled, highly reproducible and relatively inexpensive animal model for studies of the virulence of L. pneumophila, and possibly, of the role of pro-inflammatory cytokine production in this phenomenon.

Five sporadic cases of nosocomial Legionnaires’ disease were documented from 1989 to 1997 in a hospital in northern Italy. Two of them, which occurred in a 75-year-old man suffering from ischemic cardiopathy and in an 8-year-old girl suffering from acute leukemia, had fatal outcomes. Legionella pneumophila serogroup 6 was isolated from both patients and from hot-water samples taken at different sites in the hospital. These facts led us to consider the possibility that a single clone of L. pneumophila serogroup 6 had persisted in the hospital environment for 8 years and had caused sporadic infections. Comparison of clinical and environmental strains by monoclonal subtyping, macrorestriction analysis (MRA), and arbitrarily primed PCR (AP-PCR) showed that the strains were clustered into three different epidemiological types, of which only two types caused infection. An excellent correspondence between the MRA and AP-PCR results was observed, with both techniques having high discriminatory powers. However, it was not possible to differentiate the isolates by means of ribotyping and analysis of rrn operon polymorphism. Environmental strains that antigenically and chromosomally matched the infecting organism were present at the time of infection in hot-water samples taken from the ward where the patients had stayed. Interpretation of the temporal sequence of events on the basis of the typing results for clinical and environmental isolates enabled the identification of the ward where the patients became infected and the modes of transmission of Legionella infection. The long-term persistence in the hot-water system of different clones of L. pneumophila serogroup 6 indicates that repeated heat-based control measures were ineffective in eradicating the organism.

Legionella pneumophila is an intracellular pathogen whose replication in macrophages is mainly controlled by IFN-gamma. Freshly isolated peritoneal macrophages elicited in vivo with thioglycolate (TG) from A/J mice are highly permissive to L. pneumophila growth in vitro, while TG-elicited macrophages from CD1 mice are resistant. In this study, we show that when CD1 TG-macrophages are cultured for 7 days, they become permissive to Legionella infection. We demonstrate that treatment with type I IFN (IFN-alphabeta) totally inhibits the growth of L. pneumophila in both freshly isolated A/J and in vitro-aged CD1 TG-macrophages. IFN-alphabeta protective effect on permissive macrophages was comparable to that induced by IFN-gamma. Even low doses of either IFN-alpha or IFN-beta alone were effective in inhibiting L. pneumophila multiplication in macrophage cultures. Notably, treatment of resistant, freshly isolated CD1 TG-macrophages with Ab to mouse IFN-alphabeta significantly enhanced their susceptibility to Legionella infection in vitro, thus implying a role of endogenous IFN-alphabeta in mediating the natural resistance of macrophages to L. pneumophila infection. Finally, addition of anti-IFN-gamma-neutralizing Ab did not restore Legionella growth in IFN-alpha- or IFN-beta-treated A/J or CD1 permissive macrophages, indicating that IFN-alphabeta effect was not mediated by IFN-gamma. This observation was further confirmed by the finding that IFN-alphabeta was effective in inhibiting L. pneumophila replication in macrophages from IFN-gamma receptor-deficient mice. Taken together, our results provide the first evidence for a role of IFN-alphabeta in the control of L. pneumophila infection in mouse models of susceptible macrophages and suggest the existence of different pathways for the control of intracellular bacteria in macrophages.