In vitro susceptibility of isolates of Borrelia burgdorferi s.l. to antimicrobial agents.

In the present study, we investigate the in vitro antimicrobial activity of macrolides, beta-lactams and tetracycline against Borrelia burgdorferi s.l. clinical and tick isolates. Minimal inhibitory concentrations (MICs) were determined in normal growth condition and after pre-exposure of the strains to sub-MIC of the founder of each drug family. All the classes of tested antibiotics showed good antibacterial activity against all the borreliae isolates and there were no significant susceptibility differences among clinical and tick isolates. After pre-exposure of the strains to sub-MIC of erythromycin, cefoxitin and tetracycline, we observed that some strains of B. burgdorferi s.l. showed higher MIC values to both the pre-exposed drug and drugs of the same family. The less susceptibility of borreliae, in the last growth condition in vitro, could be one of the justifications of clinical results indicating the limited efficacy of these antibiotics in treatment of B. burgdoferi infections.

Prevalence and incidence of antibodies to Borrelia burgdorferi and to tick-borne encephalitis virus in agricultural and forestry workers from Tuscany, Italy.

The ticks Ixodes persulcatus and Ixodes ricinus are the main vectors of both Borrelia burgdorferi sensu lato and tick-borne encephalitis (TBE) virus in Eurasia. Borrelia burgdorferi is the cause of Lyme borreliosis, and TBE is a biphasic meningoencephalitis induced by an arbovirus belonging to the flavivirus family. The principal aims of the current investigation were (i) to determine the frequency of serological evidence of Borrelia burgdorferi sensu lato and TBE infections in healthy agricultural and forestry workers, (ii) to determine the incidence of seroconversion for antibodies against Borrelia burgdorferi sensu lato and TBE virus in Tuscan workers during a 1-year survey; and (iii) to assess the occupational risk for agricultural and forestry activities in a defined area (Tuscany, Italy). A total of 412 blood samples were taken from agricultural and forestry workers, and information on age, duration of employment, and history of tick bites was collected in a questionnaire to establish the risk factors for the diseases. Three hundred sixty-five blood donors from the same region served as controls. To estimate the rate of seroconversion, 176 of the agricultural and forestry workers were tested 1 year later. IgG and IgM antibodies against Borrelia burgdorferi sensu lato and TBE virus were detected in serum by an enzyme-linked immunosorbent assay and confirmed by Western blot analysis for Borrelia burgdorferi and by a test for inhibition of hemagglutination for TBE. Antibodies against Borrelia burgdorferi were more frequent among the workers than in the control group (7.8% vs. 4.9% in the IgG-IgM enzyme-linked immunosorbent assay and 7.03% vs. 3.56% in the confirmatory test). No seropositivity was observed for TBE virus. Eighteen of 176 subjects who underwent a second blood test developed specific antibodies against Borrelia burgdorferi within 1 year.

Molecular characterization of first human Bartonella strain isolated in Italy.

The aim of this study was to characterize a Bartonella strain (BA-1) isolated from a blood culture of an Italian, human immunodeficiency virus-positive patient with bacillary angiomatosis. We analyzed the isolate using molecular biology methods such as whole-cell fatty acid analysis, PCR-restriction fragment length polymorphism analysis, type-specific 16S rRNA PCRs, sequence analysis of the 16S rRNA, pulsed-field gel electrophoresis, and arbitrarily primed PCR. The BA-1 isolate turned out to be a Bartonella quintana strain, similar but not identical to B. quintana Oklahoma, which was used as a control strain.

Leptospira strains kept at the National Centre for Leptospirosis in Rome, Italy.

Since the National Centre for Leptospirosis (Department of Bacteriology and Medical Mycology, Istituto Superiore di Sanità, Rome) was established in 1956 by B. Babudieri, efforts have been devoted to identifying new Leptospira isolates and maintaining a collection of strains that today comprises 670 strains, 550 of which have been totally or partially classified, and 120 are still under study. This collection includes 23 serogroups and 156 serovars of pathogenic leptospires, and 32 serogroups and 54 serovars of saprophytic leptospires. The conventional serogroup and serovar identification, mainly based on antigenic relatedness, is tedious and time-consuming, requiring the maintenance of a comprehensive collection of serovar reference strains and the preparation of the corresponding rabbit antisera. Although considerable difficulties are encountered in the classification of leptospires at the serogroup and serovar level, this classification system is essential to obtain information on the epidemiology of leptospirosis in the different geographical areas. Serovar identification has become faster with the introduction of pulsed-field gel electrophoresis (PFGE) of large DNA fragments obtained after digestion of leptospiral DNAs with rare-cutting restriction enzymes. This technique has been successfully utilized to discriminate between closely related serovars of the Leptospira interrogans complex. We have recently used PFGE to characterize several Italian leptospiral isolates, confirming that PFGE analysis combined with microscopic agglutination test (MAT) with monoclonal and polyclonal antibodies can be used as an accurate and reliable method to compare and classify leptospires.

Isolation and characterization of Borrelia burgdorferi sensu lato strains in an area of Italy where Lyme borreliosis is endemic.

Between 1993 and 1998, we isolated Borrelia burgdorferi sensu lato from 55 of the 119 patients with clinically diagnosed Lyme borreliosis who were admitted to "San Martino" Hospital in Belluno, Veneto, an Adriatic region in northeastern Italy where Lyme borreliosis is endemic. Upon hospitalization, all patients presented erythema migrans. Isolates were typed using ribosomal DNA PCR-restriction fragment length polymorphism (RFLP) analysis of the rrfA-rrlB intergenic spacer. Of the 41 isolates typed, 37 belonged to Borrelia afzelii, 2 to Borrelia garinii, and 2 to B. burgdorferi sensu stricto. Pulsed-field gel electrophoresis, performed on 21 strains (13 new isolates and 8 controls), revealed different RFLP patterns within the B. garinii and B. afzelii strains; among the five B. garinii strains and the 12 B. afzelii strains, three or two different RFLP patterns were identified, according to the restriction enzyme used. The protein patterns of the new isolates confirmed their genotypic classification and revealed the level of expression of some immunodominant proteins like OspA and other characteristic Osps. These findings constitute the first report of such a high recovery rate of B. burgdorferi from patients in a very restricted area in Italy; they also indicate the predominance of the genospecies B. afzelii in the study area and the heterogeneity of the circulating strains.

Prevalence of antibodies to Leptospira serovars in sheep and goats in Alto Adige-South Tyrol.

Serum samples from 313 sheep and 95 goats were collected during November 1993 in 26 localities in Alto Adige-South Tyrol and tested by microscopic agglutination test for antibodies to 28 serovars of the genus Leptospira. At the time of blood collection all the animals appeared healthy with no clinical sign suggestive of leptospirosis. The observed seroprevalence in sheep was 6.1%, whereas the seropositivity rate for goat serum samples was 2.1%. The highest serological prevalence in sheep was recorded for serovar castellonis, followed by poi, sejroe, hardjo subtype hardjobovis, copenhageni, and cynopteri. Titres to poi were the only ones found in goats. These findings, which are proof of Leptospira infection in Alto Adige-South Tyrol, indicate that foci of several serovars exist in this region.

Epidemiological trend of human leptospirosis in Italy between 1994 and 1996.

In the three-year period 1994 1996, 222 reports on human cases of leptospirosis were received by the Italian Ministry of Health. The average annual number of reports was 29.2% lower than in the preceding eight years. In all cases but two the infections were thought to have been acquired in Italy. As in previous years, the majority of cases was observed in the northern regions of the country (83.8%), mostly in males (88.9%). Cases occurred in all age groups, but were more common in the working-age population (15-64 years). There was no common-source outbreaks. The typical leptospiral seasonal course, with a peak in August, was observed. During 1994, leptospirosis was the reported cause of death in 19 patients. Mortality was higher among males than females. The overall fatality rate was 22.6%. During the study period, a total of 126 cases of leptospirosis were confirmed by the National Centre for Leptospirosis or one of the 12 Regional Leptospira Laboratories. Of the 103 patients for whom information on place of residence, contact with animals, occupational and recreational activities was available, 98 (95.1%) were people who live in rural areas or devote themselves to occupational or recreational activities at risk. The likely source of infection and the mode of exposure were known for 55 patients. Forty-five patients (81.8%) were likely infected by contaminating water (43 cases) or soil (2 cases), ten (18.2%) by direct contact with animals or animal urine. Both running (51.2%) and stagnant water (27.9%) have been reported as a source of infection. Rodents were implicated in 50.0% of the 10 cases involving animals. In comparison with the preceding eight-year period, the risk of contracting leptospirosis was found to have increased for recreational activities (from 34.7 to 38.2%) and decreased for occupational activities (from 45.8 to 32.7%). A large number of infections, however, was ascribed to accidental events (25.5%). As in the previous period, besides fever, the involvement of the liver was the most frequent clinical manifestation (70.8%). Influenza-like symptoms were the only signs of illness in 15.1% of cases. Infections by 9 different serogroups were detected. The most frequent antibodies were those against serovars icterohaemorrhagiae, poi, copenhageni and brattislava. The presence of co-agglutinins against serovars belonging to different serogroups prevented the identification of the presumptive infecting serogroup in 19.8% of subjects.

Legionnaires' disease on a cruise ship linked to the water supply system: clinical and public health implications.

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.

Lyme disease in Italy, 1983-1996.

This paper is a brief review of the epidemiology of Lyme disease in Italy. The first case of the illness was identified by Crovato in Liguria in 1983. In the following years, many other cases have been reported from all Italian regions with the exception of Valle d'Aosta, Basilicata and Calabria. The exact number of cases in our country is not known because Lyme disease was not a notifiable disease until 1990, but on the basis of literature data, at least 1324 cases have been observed in the fourteen-year period 1983-1996. Friuli-Venezia Giulia, Veneto and Trentino-Alto Adige are the main regions involved. Only few cases of illness have been described in Mid and Southern Italy and in the Islands (6.0%). No reports exist on Lyme disease in animals. There is, however, serological evidence of infection of domestic and wild animals. The causative agent, Borrelia burgdorferi sensu lato, was first isolated from Ixodes ricinus ticks by Cinco in Trieste in 1977. Since then many other strains, belonging to three different genomic species (B. burgdorferi sensu stricto, B. garinii and B. afzelii), have been isolated from humans, reservoir hosts and ticks. Cases were reported for all age-groups, more frequently in females, following the typical seasonal course, with a marked seasonality from spring to autumn, when ticks are more active. Erythema chronicum migrans was the most frequent manifestation of LD. Several studies have been conducted on groups at risk (forest workers, gamekeepers, etc.). In contrast to the high prevalence of antibodies to B. burgdorferi sensu lato in the groups at risk (up to 27.2% for forest workers), the seroprevalence of the healthy population is, in general, lower.

Antigenic and genomic analysis of a Borrelia burgdorferi sensu stricto strain isolated from Ixodes ricinus ticks in Alto Adige-South Tyrol, Italy.

A Borrelia burgdorferi sensu lato strain isolated from IXodes ricinus ticks in Alto Adige-South Tyrol (Northern Italy) was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole cell proteins, Western immunoblotting analysis (WBA) with polyclonal and monoclonal antibodies, and pulsed-field gel electrophoresis (PFGE). The isolate named BZ6 was identified as belonging to the genospecies B. burgdorferi sensu stricto on the basis of its protein profile and its reactivity with monoclonal and polyclonal antibodies. The PFGE study performed with the two rare-cutting restriction enzymes MluI and SmaI confirmed the SDS-PAGE and WBA characterizations, but showed a genetic diversity between the isolate and two out of the three B. burgdorferi sensu stricto strains used in this study as controls, the American type strain B31 and the locally isolated strain BZ1. No difference in the PFGE patterns between the isolate BZ6 and the Swiss strain IRS was noted. Our findings show the value of PFGE analysis for classifying B. burgdorferi sensu lato isolates and for revealing their genetic diversity, and its usefulness for epidemiological investigations.

The complement-killing of Borrelia burgdorferi. Target antigens and sensitizing antibodies.

It had been previously shown by the Microbial Adherence Immobilization Assay (MAIA) that Borrelia burgdorferi sensu stricto, type strain B31 was clumped, immobilized and killed in vitro by sensitizing antibodies that activated the classical complement pathway and the complement-killing of live borrelia. In the present study, the target antigens and sensitizing antibodies responsible for the complement-killing of borrelia were investigated, using MAIA as a selective identification tool. It was found that the fractions containing the 31 and 34 kDa outer surface proteins from strain B31 were the unique antigens producing sensitizing antibodies in rabbits that activated the complement-killing of B31. An anti-OspB, but not an anti-OspA, monoclonal antibody did activate the B31 complement-killing in MAIA. From these results, constraints on the effectiveness of OspB and OspA as immunogens for the prevention and control of Lyme borreliosis in humans are discussed.

Serologic survey for antibodies to Borrelia burgdorferi in sheep, goats and dogs in Cordillera Province, Bolivia.

A serosurvey for antibodies to Borrelia burgdorferi using an enzyme-linked immunosorbent assay (ELISA) was conducted on sheep, goat and dog serum samples collected in Cordillera Province, Bolivia, in 1992 Sera from 98 sheep, 218 goats and 43 dogs were tested. The observed seroprevalence in sheep and dogs was 0.0%, whereas the seropositivity rate for goat serum samples was 5.0%. Upon analysing 10 positive sera by Western immunoblotting, five reacted against the specific protein antigens and all of them met the criteria for positivity on the basis of immunoglobulin G (IgG) bands, indicating that goats in Cordillera Province were exposed to B. burgdorferi. These findings, which are further proof of the existence of B. burgdorferi infection in Bolivia, indicate the serologic analysis of goats as a suitable tool for Lyme borreliosis surveillance.

Serological survey of leptospiral infections in sheep, goats and dogs in Cordillera province, Bolivta.

A serological survey for antibodies to Leptospira spp. was conducted on sheep, goat and dog serum samples collected in three localities in Cordillera province in the southern part of the Santa Cruz Department (Bolivia) in 1992. A total of 98 sheep, 218 goats and 43 dogs were tested against 29 leptospiral serovars using the microscopic agglutination test. At the time of blood collection all of the examined animals appeared healthy and presented no clinical sign suggestive of leptospirosis. Antibody prevalences, as determined by positive results at a 1:100 dilution or higher, was 14.3% in sheep, 19.7% in goats, and 14.0% in dogs. Agglutinins against six serovars (poi. shermani, pomona, canicola, javanica, djasiman) were found in positive animals. The highest serological prevalence in sheep and goats was recorded for serovar poi, followed by pomona in sheep and shermani in goats. Titres to shermani were the commonest in dogs. The results of this survey indicate that leptospiral infection is common in south-east Bolivia and that serovars of several serogroups concur in the etiology.

Molecular epidemiology of an outbreak of Legionnaires' disease associated with a cooling tower in Genova-Sestri Ponente, Italy.

Fatty acid profile analysis, monoclonal antibody (MAb) subtyping, pulsed-field gel electrophoresis (PFGE), arbitrarily primed polymerase chain reaction (AP-PCR), and ribotyping were used to compare clinical and environmental Legionella pneumophila serogroup 1 isolates from an outbreak of Legionnaires' disease presumptively associated with cooling towers. According to the Oxford subtyping scheme, the MAb subtype of patients' isolates and of two strains originating from a cooling tower was Pontiac, whereas the other isolates were subtype Olda. The strains showed no intrinsic strain-to-strain difference in fatty acid profiles, and ribotyping and length polymorphism of the 16S-23S rDNA intervening regions failed to reveal any differences between the isolates. Conversely, PFGE and AP-PCR appeared to be more discriminatory, as the same genomic profile was found for the clinical and some environmental strains. Meteorologic and epidemiological data and the results of molecular analysis of the Legionella pneumophila serogroup 1 isolates support the hypothesis that the infection was transmitted from one of the cooling towers to the indoor environment of the same building, to homes in proximity that had open windows, and to the streets. In fact, the outbreak diminished and later ended after a part in the tower was replaced. This investigation demonstrates the utility of combined molecular methods (i.e., phenotypic and genomic typing) in comparing epidemiologically linked clinical and environmental isolates. Finally, the outbreak confirms the risk of Legionnaires' disease posed by cooling towers, mainly when atmospheric thermal and humidity inversions occur. This finding emphasizes the need to determine whether the source of infection is in the living or working environment or somewhere else.

Antibodies to Borrelia burgdorferi in sheep and goats. Alto Adige-South Tyrol, Italy.

A serologic survey for antibodies to Borrelia burgdorferi was conducted on sheep and goat serum samples collected in Alto Adige-South Tyrol, Italy, in 1990. Sera were tested by Indirect Immune Fluorescence Assay (IIFA) and Microbial Adherence Immobilization Assay (MAIA). IIFA and/or MAIA anti-B. burgdorferi antibodies were detected in 14.1% of the 269 sheep and 36.8% of the 133 goats examined. IIFA and MAIA were both positive in 4 out of 38 positive sheep sera (10.5%) and 21 out of 49 positive goat sera (42.8%). These discrepancies suggest that MAIA- and IIFA-detected antibodies do differ from each other. The detection by MAIA of antibodies sensitizing B. burgdorferi to the killing effect of complement seems to be a valid parameter to evaluate the acquired immunity of sheep and goats to B. burgdorferi infections.

Human leptospirosis in Italy, 1986-1993.

In the eight-year period 1986-1993, the Italian National Center for Leptospirosis and the Regional Leptospira Laboratories confirmed 312 cases of clinical leptospirosis by using the microscopic agglutination (MA) assay. The majority of cases was observed in Northern regions of the Country. Cases were reported in all age groups, but were most common in the working-age population. Of 312 cases, 291 (93.3%) occurred among males. The largest number of infections was ascribed to occupational activities (45.8%). The typical leptospiral seasonal course, with a peak during the summer, was observed. Involvement of the liver was the most frequent manifestation. Influenza-like symptoms were the only signs of illness in 11.1% of cases. Anti-leptospira antibodies, cross-reacting with two or more serovars, were found in 28.2% of sera. The most frequent serovar-specific antibodies were those against poi, icterohaemorrhagiae, bratislava, copenhageni and sejroe.

Genetic and phenotypic characterization of Borrelia burgdorferi strains isolated from Ixodes ricinus ticks in the Province of Bolzano, Italy.

Lyme disease is caused by three borrelial species, B. burgdorferi sensu stricto, B. garinii and Borrelia group VS461. In a restricted biotope of the Bolzano province, in the Caldaro community, five clones of two borrelial variants were isolated from Ixodes ricinus ticks. A preliminary serological study showed that the two variants cross-reacted with B. burgdorferi B31 and B. garinii N34 strains, respectively. The isolates were genomically related with strains B31 and N34, respectively, sharing a similar plasmid and restriction fragment length polymorphism profile with these strains. The phenotypic pattern of the Caldaro isolates-namely their protein and antigenic profile-showed infra-subspecific variation compared to related strains B31 and N34 respectively. The observed phenotypic variability between strains isolated from the same biotope and in the same tick host strongly indicated the variability of gene-encoded characters is a constant characteristic of borrelial strains, even when from the same ecological niche.