Prevalence, types, and geographical distribution of Listeria monocytogenes from a survey of retail Queso Fresco and associated cheese processing plants and dairy farms in Sonora, Mexico.

In the first part of this study, samples were collected from farms, cheese processing plants (CPPs), and retail markets located in various geographical areas of Sonora, Mexico, over a 12-month period during the summer of 2004 and winter of 2005. Four (all Queso Fresco [QF] from retail markets) of 349 total samples tested positive for Listeria monocytogenes (Lm). Of these four positive samples, three were collected in the northern region and one in the southern region of Sonora. Additionally, two were collected during the winter months, and two were collected during the summer months. For the second part of the study, a total of 39 samples from a farm, a CPP, and retail markets were collected and processed according to a combination of the Norma Oficial Mexicana NOM-143-SSA1-1995.10 method (NOM) and the U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual method, and 27 samples from these same locations were collected and processed according to the U.S. Department of Agriculture Food Safety and Inspection Service method (USDA-FSIS). The NOM-FDA method recovered the pathogen from 6 (15%) of 39 samples (one cheese and five product contact surfaces), while the USDA-FSIS method recovered the pathogen from 5 (18.5%) of 27 samples (all product contact surfaces). In addition, the 40 isolates recovered from the 15 total samples that tested positive for Lm grouped into five distinct pulsotypes that were ca. 60% related, as determined by pulsed-field gel electrophoresis analysis. The results of this study confirmed a 3.4% prevalence of Lm in QF collected from retail markets located in Sonora and no appreciable difference in the effectiveness of either the NOM-FDA or USDA-FSIS method to recover the pathogen from cheese or environmental samples.

Serological evidence of Rickettsia infections in forestry rangers in north-eastern Italy.

The prevalence of antibodies to Rickettsiae and other tick-borne microrganisms in the sera of 181 forestry rangers from Friuli-Venezia-Giulia, Italy, was examined. Seven (3.9%) sera were positive for Rickettsia conorii and Rickettsia helvetica, as single or dual infections; four of these sera had been found previously to be positive for Borrelia burgdorferi. Antibodies to Coxiella burnetii were detected in five (2.8%) sera, four of which were also positive for B. burgdorferi. These findings indicate that patients in this north-eastern Italian region with fever subsequent to tick-bite should be investigated for Rickettsia and Coxiella infections.

Leptospirosis following a flood in the Veneto area, North-east Italy.

In August 2002, an exceptional flood devastated a suburban area in the surroundings of Vicenza, North-east Italy. A fatal case of haemorrhagic pneumonia, which was presumptively diagnosed as leptospirosis, was observed as a consequence of the inundation. A local seroepidemiological survey was activated thereafter, with the principal aim of evaluating the risk of infection with Leptospirae in the population exposed to the flood. A 6.8% seroconversion rate was found in the population studied; however, the case previously observed remained unique, since an overt outbreak of leptospirosis did not occur.

Induction of cystic forms by different stress conditions in Borrelia burgdorferi.

Cystic forms of Borrelia burgdorferi might represent a low metabolic activity state or phase of B. burgdorferi cells that allows the spirochete to survive in a hostile environment until conditions are favourable to multiply again. In this study we evaluated the rate of cyst formation induced by oxidative stress, pH variations, and heating, reconversion of cysts to vegetative forms, and some aspects of their metabolic activity. We observed cyst formation in the presence of extreme pH values, and at high temperature, but the best production of cystic forms was observed in the presence of H2O2. When transferred to BSK II medium, the cystic forms reconverted to spirochetes in relation to their age and type of induction treatment. Furthermore, we demonstrated a low metabolic activity of cystic forms by measuring amino acid incorporation. Overall, these data suggest that the phenomenon of conversion to cysts by B. burgdorferi provides a limited survival potential. This short-term survival, however, gives borreliae an additional chance to overcome unfavourable environmental conditions.

Conversion of Borrelia garinii cystic forms to motile spirochetes in vivo.

Cystic forms (also called spheroplasts or starvation forms) and their ability to reconvert into normal motile spirochetes have already been demonstrated in the Borrelia burgdorferi sensu lato complex. The aim of this study was to determine whether motile B. garinii could develop from cystic forms, not only in vitro but also in vivo, in cyst-inoculated mice. The cysts prepared in distilled water were able to reconvert into normal motile spirochetes at any time during in vitro experiments, lasting one month, even after freeze-thawing of the cysts. Motile spirochetes were successfully isolated from 2 out of 15 mice inoculated intraperitoneally with cystic forms, showing the infectivity of the cysts. The demonstrated capacity of the cysts to reconvert into motile spirochetes in vivo and their surprising resistance to adverse environmental conditions should lead to further studies on the role and function of these forms in Lyme disease.

Characterization of the first tick isolate of Borrelia burgdorferi from Italy.

We report on the first isolation of a spirochetal organism from Ixodes ricinus ticks of the Trieste area (Northern Italy) which was identified as Borrelia burgdorferi by its reactivity with specific monoclonal antibodies directed against the OSPA and flagella proteins.

Selection of a Borrelia burgdorferi antigenic variant by cultivation in the presence of increasing amounts of homologous immune serum.

This investigation was undertaken to select antigenic variants of a Borrelia burgdorferi strain in vitro. The original strain BITS was cultivated in BSK medium supplemented with increasing concentrations of homologous hyperimmune serum raised in rabbits. After a few serial passages starting from a subinhibitory serum dilution of 1:800 in BSK up to 1:200, a variant named BITSv was obtained; it grew abundantly like the control culture in the presence of hyperimmune serum. Analysis of the antigenic pattern of the original and derived variants by Western blotting revealed that BITSv, compared to the original strain BITS, had lost the reactivity with the immune serum at the level of the oligosaccharide moiety. These experiments, designed to mimic the possible action of antibodies that arise during a Borrelia infection, suggest that lipopolysaccharides are surface located and that they play a role in the integrity of the outer membrane during the multiplication of Borrelia burgdorferi.

Evidence of Dbps (decorin binding proteins) among European strains of Borrelia burgdorferi sensu lato and in the immune response of LB patient sera.

Decorin binding proteins DbpA and DbpB act as Borrelia burgdorferi (B. burgdorferi) adhesins to decorin, and are able to elicit a persistent antibody response in the mouse; accordingly DbpA protein would seem to be promising in immunoprofilaxis of Lyme borreliosis (LB). This study examines the distribution of Dbp epitopes in European strains of B. burgdorferi, of different genospecies and the presence of antibodies to Dbps in human sera from patients suffering from early and late LB, as revealed by immunoblotting. Different levels of expression of Dbp epitopes were found both among and within genospecies; data from human sera indicate that Dbps are expressed during infection though not as strongly as in the mouse infection.

Oligonucleotides specific for pathogenic and saprophytic leptospira occurring in water.

Sets of primers specific for both pathogenic (SPL) and saprophytic (SSL) Leptospira were designed from ribosomal 16S genes (rrs) available in databases. They were used as two sets of primer pairs for the PCR amplification of known pathogenic and saprophytic strains. It was possible to identify pathogenic strains by the use of SPL primers and saprophytic ones by SSL primers. Serovars from L. meyeri, of controversial pathogenicity status, confirmed the heterogeneity of the species representatives in this respect. Serovars ranarum, sofia and perameles were amplified by SPL and not SSL. Conversely, serovar semaranga was amplified by SSL and not SPL. In order to use SPL primers for the detection of pathogenic leptospires from a natural water environment, we set up an additional semi-nested PCR by employing a second internal primer which succeeded in detecting as few as 5 pathogenic leptospires per ml of water.

Simultaneous measurement by flow cytometry of phagocytosis and metabolic burst induced in phagocytic cells in whole blood by Borrelia burgdorferi.

This paper describes the interactions between a strain of Borrelia burgdorferi and phagocytic cells, measured in whole blood, by a two-color flow cytometric method, which allowed the simultaneous quantification of both the phagocytosis rate and the oxidative burst activation. The data obtained indicated that: a) phagocytosis and metabolic activation increased as a function of spirochete concentration; b) the number of ingesting cells peaked within 10 min but activation followed later, and did not involve all the phagocytosing cells; c) opsonization of borreliae with a patient's serum enhanced the two cellular activities, mostly phagocytosis. The intensity of such functions was lower than those found for Staphylococcus aureus. The flow cytometric assay of phagocytes interactions with Borrelia burgdorferi assessed in whole blood represents an experimental approach which simulates the physiological conditions in nature.

Leptospira interrogans and Leptospira peptidoglycans induce the release of tumor necrosis factor alpha from human monocytes.

Elevated plasma concentrations of the cytokine tumor necrosis factor alpha (TNF alpha) have been observed in patients affected by leptospirosis. In this study we found that a preparation of peptidoglycan of Leptospira interrogans, serovar copenhageni, was able to induce the release of TNF alpha from peripheral blood mononuclear cells. TNF alpha induction occurred in a dose dependent manner and was not affected by the endotoxin inhibitor polymixin B. This is the first report on induction of TNF alpha release by a peptidoglycan of spirochetes. Our findings are consistent with existing clinical data and provide a potential mechanism for TNF alpha production.

Seroprevalence of tick-borne infections in forestry rangers from northeastern Italy.

The aim of this study was to estimate the seroprevalence of antibodies to Borrelia burgdorferi, Anaplasma phagocitophilum and tick-borne encephalitis (TBE) virus, and risk factors, in forestry rangers from the Friuli-Venezia-Giulia region in northeastern Italy. Sera from 181 forestry rangers were examined with two-tiered serological tests for TBE, Lyme borreliosis and ehrlichiosis. Information about risk factors such as job location, residence, number of tick bites and outdoor leisure activities was collected with a questionnaire. Seropositivity was 0.6% for TBE virus, 23.2% for Lyme borreliosis and 0.6% for ehrlichiosis. Lyme borreliosis positivity, as determined by Western blot, was associated with working in the foothills, with gardening in the northeastern part of the region, and with a history of yearly tick bites. Risk factors were similar when a case of Lyme borreliosis was defined either by Western blot positivity or by clinical history.

Guidelines for the diagnosis of tick-borne bacterial diseases in Europe.

Ticks are obligate haematophagous acarines that parasitise every class of vertebrate (including man) and have a worldwide distribution. An increasing awareness of tick-borne diseases among clinicians and scientific researchers has led to the recent description of a number of emerging tick-borne bacterial diseases. Since the identification of Borrelia burgdorferi as the agent of Lyme disease in 1982, 11 tick-borne human bacterial pathogens have been described in Europe. Aetiological diagnosis of tick-transmitted diseases is often difficult and relies on specialised laboratories using very specific tools. Interpretation of laboratory data is very important in order to establish the diagnosis. These guidelines aim to help clinicians and microbiologists in diagnosing infection transmitted by tick bites and to provide the scientific and medical community with a better understanding of these infectious diseases.

Prevalence of IgG reactivity in Lyme borreliosis patients versus Borrelia garinii and Borrelia afzelii in a restricted area of Northern Italy.

This survey evaluates the antibody band patterns of sera taken from clinically defined cases of Lyme borreliosis, towards three locally isolated strains of Borrelia burgdorferi, belonging to the three species: Borrelia sensu stricto, Borrelia garinii and Borrelia afzelii, by means of Western blot. The sera were taken from patients resident in a limited area of Friuli Venezia Giulia (FVG) region. The data indicated that, besides a different feature of the band reactivity which correlated to the different stages of Lyme borreliosis, there was a preferential reactivity to the species Borrelia afzelii and Borrelia garinii. An immunodominant band at 51 kDa, corresponding to a protein visible in the electrophoretic profile of strain BL3 (B. afzelii), behaved like a marker of an early infection, because it was present exclusively in the sera of patient with ECM. The overall findings would indicate that B. afzelii and B. garinii are the prevalent genospecies in the FVG area, even if strains belonging to B. sensu stricto have been also isolated in this area. Consequently strains representative of these two species must be used as antigens in Western blot.

IgM and IgG significant reactivity to Borrelia burgdorferi sensu stricto, Borrelia garinii and Borrelia afzelii among Italian patients affected by Lyme arthritis or neuroborreliosis.

This survey evaluates the specificity of band patterns in immunoblot of sera taken from clinically defined cases of Lyme arthritis and neuroborreliosis, towards three locally isolated strains of Borrelia burgdorferi, belonging to the three species: Borrelia sensu stricto, Borrelia garinii and Borrelia afzelii. To assess specificity, patient sera were statistically (X2, P < or = 0.05) compared with blood donors sera samples. Both IgG and IgM antibodies were considered. The overall reactivity of the three Borrelia strains in IgG immunoblots indicated that ten protein bands were significant, with a different prevalence of some of them in the two groups of patient sera: bands at 60-58, 30-33, 36-37 and 28-27 kDa were markers for neuroborreliosis sera; proteins at 100-83, 72-70 and 18-17 kDa behaved like markers for Lyme arthritis. The IgM Immunoblots revealed significant bands at 100-83, 72-70, 51, 24-21 and 18-17 kDa only with neuroborreliosis sera. Though there were variable band reactivities in each strain, a correlation emerged between the three genospecies and the clinical symptoms: in fact B. afzelii and B. garinii were prevalent in Lyme arthritis sera, (IgG Immunoblots); B. garinii was associated to neuroborreliosis (IgG and IgM Immunoblots); B. sensu stricto was strongly reactive with neuroborreliosis in IgM immunoblots. These data indicate that the three locally strains of Borrelia representing the three genospecies should be used together in immunoblot to detect antibodies elicited in neuroborreliosis and Lyme arthritis.

Identification of Leptospira biflexa by real-time homogeneous detection of rapid cycle PCR product.

Sequence analysis of 16S rRNA genes extracted from nucleic acids databases enabled the identification of a Leptospira biflexa (L. biflexa) signature sequence, against which a reverse primer designated L613, was designed. This primer, when used in conjunction with a universal bacterial specific forward primer designated Fd1, enabled the development of a LightCycler-based PCR protocol in which fluorescence emission due to binding of SYBR Green I dye to amplified products could be detected and monitored. A melting temperature (Tm), determined from the melting curve of the amplified product immediately following the termination of thermal cycling, confirmed that the product was that of L. biflexa. Agarose gel electrophoresis therefore was not necessary for identification of PCR products. The PCR protocol was very rapid, and consisted of 30 cycles with a duration of 20 s for each cycle with the monitoring of the melting curve requiring an additional 3 min. The whole protocol was completed in less than 20 min. The PCR protocol was also specific and enabled the identification of 18 strains of L. biflexa, whilst excluding 14 strains of L. interrogans and Leptonema illini. Two examples of its utility in improving work flow of a Leptospira reference laboratory are presented in this article. The use of a simple boiling method for extraction of DNA from all the members of the Leptospiraceae family DNA further simplifies the procedure and makes its use conducive to diagnostic laboratories.

Tick-borne borrelioses pathogen identification in Ixodes ticks (Acarina, Ixodidae) collected in St. Petersburg and Kaliningrad Baltic regions of Russia.

Two isolated Baltic seashore populations of Ixodes ticks were studied as vectors of different Borrelia genospecies in Russia by using darkfield microscopy and modified polymerase chain reaction (PCR). In the Kalinigrad region (Kurish Spit, forests near the settlements of Lesnoye and Rybachy), 788 Ixodes ricinus (L.) adults and nymphs were collected by flagging and studied by darkfield microscopy during 1995-1996. There were 88 darkfield microscopy positive specimens (11.2%) of which 69 were also analyzed by PCR. Borrelia afzelii and B. garinii were found individually and together in ticks. In this region, on the Kurish Spit, 7 patients with tick borrelioses were observed: 2 in the Russian part of Spit and 5 in the Lithuanian part. A significant difference was found between Borrelia prevalence during the spring and fall peaks of tick abundance. Specimens that were darkfield microscopy positive prevailed in the fall (25.15%) in comparison with the spring peak (7.3%). The number of specimens with identified genospecies prevailed in the spring: 22 out of 35 versus 4 out of 31 in the fall. Among 29 PCR positive I. ricinus, 21 contained B. afzelii, 3 had B. garinii, and 2 had dual infection. In 1995, only B. afzelii infected specimens were observed. In the vicinity of St. Petersburg (the seashore of the northern Gulf of Finland, in forests near Lisy Nos, Morskaja) during 1992-1996, 31 patients with a tick-borne borrelioses were registered. We collected 487 Ixodes persulcatus Schulze by flagging and studied them by darkfield microscopy in 1995-1996 of which 144 ticks (29.6%) were darkfield microscopy positive. Sixty darkfield-positive specimens were analyzed by PCR, and in 88.3% of cases genospecies were identified. B. afzelii and B. garinii were identified individually and together in ticks. In 1995, I. persulcatus with dual infection prevailed with 11 out of 21 (52.4% positive), whereas in 1996, most I. persulcatus ticks contained B. garinii (81.2%). Dual infection was observed in 4 of 32 (12.5%) ticks. Dual infections in I. persulcatus females increased within the seasonal peak of tick activity as was observed in 1995 and in 1996. Many patients not only had erythema migrans, but also exhibited early neurological symptoms that coincided with the number of tick vectors that had dual infections in June, indicating that these patients were bitten by female ticks that had dual infections. A significant difference existed between levels of infection in I. ricinus and I. persulcatus, with all 3 types of Borrelia infection observed 2 times more often in I. persulcatus than in I. ricinus and dual infection occurred in I. persulcatus 3.7 times more often. It appeared that I. persulcatus is a much more dangerous vector of tick-borne borrelioses than I. ricinus.

Sensitivity in vitro of leptospira to oxamicetin.

Oxamicetin was tested against 12 strains of parasitic leptospira each representing a different serotype and serogroup, and 8 saprophytic strains in liquid media. The results showed general susceptibility of parasitic leptospira being MIC's from 0.1 to 0.5 mug/ml. The MIC's against saprophytic strains were 10 similar to 40 mug/ml.

Preliminary evaluation of in-vitro antimycobacterial properties of N1-(aryliden)-2-pyridinecarboxyamidrazones.

After preliminary in vitro screening of 17 newly synthesized compounds belonging to the chemical class of N1-(aryliden)-2-pyridinecarboxyamidrazones, active against Mycobacterium tuberculosis H37Rv, the minimum inhibitory concentrations (MICs) of the ten most promising agents against three clinical isolates were determined by agar dilution. Compounds 12 and 14 were the most active, each inhibiting strain H37Rv at concentrations of 8 microg/ml and having a MIC of 16 microg/ml against the human isolates. The results obtained in this preliminary study confirmed the interesting antitubercular properties of these newly synthesized compounds and allowed us to carry out our investigations over a large number of isolated clinical strains.

Sensitivity of Borrelia and Leptospira to Quinupristin-dalfopristin (Synercid) in vitro.

In vitro activity of Quinupristin-dalfopristin (Synercid) against seventeen isolates of Borrelia burgdorferi and two representatives of Leptospira spp. was investigated. MICs ranged from 0.03 to 0.125 for B. burgdorferi and 0.125-0.25 microg/ml for Leptospires. Time killing studies carried out with 2 MIC demonstrated U 3 log(10)-unit killing after 72 h, showing a significant activity against spirochetes, though at a lower level than other antibiotics in use in the therapy of Lyme disease and leptospirosis.