Beniamino Cacciapuoti

Survey on the prevalence of leptospira infections in the Italian population

This investigation is the first nationwide survey on the circulation of leptospira infections in human beings in Italy. In nine out of twenty Italian regions, representative samples of the population were investigated for the presence of leptospira infections. Unexpectedly, leptospira infections were found to be widespread, the number of cases being much higher than the diagnosed clinical cases. There were found to be high, medium, and low risk areas. On the whole, the risk for the rural population was no higher than the risk for urban dwellers; leisure activities, contact with animals and residence on the plain versus residence in the hills were important risk factors. There was an unidentified risk factor in urbanites which was absent in the rural population. A changing pattern in infecting serovars was observed, with infections from serogroups Sejroe, Javanica and Australis prevailing over infections from the Icterohaemorrhagiae and Bataviae serogroups, which were the main agents of human leptospirosis during the 1950s. The mechanisms of these changes, the need for epidemiological surveys and improved diagnostic methods of screening are discussed.

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 againstpoi, icterohaemorrhagiae, bratislava, copenhageni andsejroe.

Fatty Acid Profiles, a Chemotaxonomic Key for the Classification of Strains of the Family Leptospiraceae

The fatty acid profiles of 59 type strains representing genera, species, and serovars belonging to the family Leptospiraceae were investigated by using gas-liquid chromatography of fatty acid methyl ester (FAME) derivatives prepared from washed leptospires. The interstrain differences of the gas-liquid chromatography FAME profiles were quantified by performing a linear regression analysis in which we compared the FAME profiles of pairs of strains and expressed the results as correlation coefficients. Leptospiral strains could be differentiated into 17 FAME relatedness groups, which were characterized by both stringent similarities within a group and sharp differences between groups. In each group, a strain was selected as a FAME reference strain. FAME reference strains can be used as keys for a chemotaxonomic phenotypic classification of strains belonging to the family Leptospiraceae.

Prevalence of leptospirosis in man. Pilot survey

A survey on the prevalence of leptospirosis was performed on the population living in an area of central Italy. The size of the sample was calculated in order to provide significant results in the case of a prevalence of infection in not less than 1 % of the population.Results demonstrated an unexpectedly wide circulation of leptospirosis in the surveyed area, showing a prevalence rate of infection of 11.34% for people living in rural areas and 3:08% for people living in the main town. The highest prevalence of infection (17.44%) was found in people between 30 and 44 years of age, living in rural areas.Such a wide circulation of undiagnosed past leptospiral infections was attributed both to the prevalence of mild clinical cases of leptospirosis in humans and the lack of microbiological tests performed to differentiate current leptospirosis from other infectious illnesses. An unexpected persistence in sera of co-agglutinins towards non-pathogenic serovars of L. biflexa was also noticed in healthy people.Criteria were established for the extension of the survey on the prevalence of leptospirosis to cover larger areas by limiting sampling to the more exposed age groups and to areas representative of a larger land belt.

Fatty Acid Profiles in the Family Leptospiraceae

Fatty acid profiles of six leptospira strains representative of genera, species, and serogroups within the family Leptospiraceae were determined by gas liquid chromatography (GLC) of fatty acid methyl ester (FAME) derivatives. The influence of methodological and biological variables on FAME profiles of the same strain was tested. FAME profiles were sharply affected by the fatty acid composition of the culture medium but not by the growth phase. Twenty-four FAME peaks were selected on the basis of their presence in repeated gas chromatographic runs of single strains. Inter-strain divergences of FAME profiles were quantified by linear regression analysis (LR). Step-wise divergences in FAME profiles were observed between strains at serogroup, species, and genus levels.

Selection of antigenic variants from Leptospira interrogans serovar canicola by means of anti-canicola monoclonal antibody.

Antigenic variants were isolated from canicola by a single selection with anti-canicola monoclonal antibody CT-3. The variants were not identical to any serovars of serogroup Canicola and thought to be a new serovar. Variation frequency was calculated at 5.9 X 10(-4). The usefulness of monoclonal antibodies for selection of antigenic variants of leptospiras is discussed.

Rickettsiae of the spotted fever group associated with the host-parasite system Oryctolagus cuniculi/Rhipicephalus pusillus.

Wild rabbits--Oryctolagus cuniculi--living in large numbers in a protected zone of Tuscany, the park of Migliarino--San Rossore--Massaciuccoli, showed to be carriers of the hard tick Rhipicephalus pusillus, previously observed in North Africa and Sicily. Antibodies to Rickettsia conorii and R. slovaca were detected in 78.9 per cent of the wild rabbits captured in that area. Seroconversion towards R. conorii was also observed in guinea pigs inoculated with homogenates of R. pusillus parasitizing the wild rabbits. These results identify an ecological niche of rickettsiae of the Spotted fever group in the host-parasite system O. cuniculi/R. pusillus. Attempts to isolate rickettsiae from the ticks and the wild rabbits were unsuccessful both in the egg yolk sac and in the guinea pig. This failure probably shows the low pathogenicity of the rickettsiae parasitizing the biosystem O. cuniculi/R. pusillus.

Chromosomal rearrangement and serovar conversion in Leptospira biflexa strains

A bacterial population change involving chromosomal rearrangement and phenotypic changes in antigens, proteins and lipopolysaccharides is described for strains of Leptospira biflexa that were previously grown in media containing homologous oligoclonal antibodies. The chromosomal rearrangement phenomenon showed that the variants differed from the parent strains, yet they were similar to phenotypically related serovars already occurring in nature. Accordingly, in vitro serovar conversion mediated by chromosomal rearrangement, due to as yet unknown genetic mechanisms, had occurred.

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.

Evaluation of a passive microcapsule agglutination test for the screening of human leptospirosis

Microcapsules, absorbed with two mixed antigens- each composed of 3 sonicated leptospira serovars — were developed in the past as a microcapsule agglutination test (MCA-LS) for the screening of clinical leptospirosis.Fof this study, fifty serum samples, taken at an earlier and at a later stage of illness from 25 Italian in-patients with clinical symptoms of leptospirosis, were tested with both the MCA-LS one-dilution test and the microscopic agglutination (MA) test, the confirmatory test for leptospirosis, with 18 leptospira strains circulating in Italy. Compared with MA, MCA-LS showed a sensitivity of 91.7%, and a specificity of 92.3%, including leptospiroses not substained by the diagnostic strains used in the MCA-LS kit.