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Consumption of raw food, especially smoked fish, meat, soft cheeses, and vegetables, contaminated with Listeria monocytogenes can cause listeriosis, which can be invasive in pregnant women, elderly...

The aim of the present study was to determine seroprevalence for Toxoplasma gondii by meat juice ELISA and evaluate the presence of T. gondii and Sarcocystis spp. within host tissues by histology, PCR and in vitro isolation, in the indigenous Cornigliese sheep breed in northern Italy. Seventeen out of 24 (70.8%) sheeps were positive for T. gondii by meat juice ELISA. Twenty sheep (83.3%) were positive by PCR for T. gondii, while 24/24 sheep (100%) were positive by PCR for Sarcocystis spp. Tissues cysts compatible with Sarcocystis spp. were visible in all animals on histology. PCR confirmed the presence of T. gondii after three weeks of in vitro culture on Vero cells in only one sample. Genotyping of T. gondii by RLFP with 5 markers showed a predominance of genotypes II/III. Sequence analysis of Sarcocystis spp. showed only the presence of Sarcocystis tenella. T. gondii and S. tenella are present in a high percentage of Cornigliese sheep in northern Italy. Future studies should concentrate upon the reproductive and economic effects of these parasitic infections, in light of the necessary conservation of this local, indigenous sheep breed.

Background Visceral leishmaniasis (VL) caused by Leishmania infantum is an ongoing health problem in southern Europe, where dogs are considered the main reservoirs of the disease. Current data point to a northward spread of VL and canine leishmaniasis (CanL) in Italy, with new foci in northern regions previously regarded as non-endemic. Methodology/Principal findings Multilocus microsatellite typing (MLMT) was performed to investigate genetic diversity and population structure of L. infantum on 55 samples from infected humans, dogs and sand flies of the E-R region between 2013 and 2017. E-R samples were compared with 10 L. infantum samples from VL cases in other Italian regions (extra E-R) and with 52 strains within the L. donovani complex. Data displayed significant microsatellite polymorphisms with low allelic heterozygosity. Forty-one unique and eight repeated MLMT profiles were recognized among the L. infantum samples from E-R, and ten unique MLMT profiles were assigned to the extra E-R samples. Bayesian analysis assigned E-R samples to two distinct populations, with further sub-structuring within each of them; all CanL samples belonged to one population, genetically related to Mediterranean MON-1 strains, while all but one VL cases as well as the isolate from the sand fly Phlebotomus perfiliewi fell under the second population. Conversely, VL samples from other Italian regions proved to be genetically similar to strains circulating in dogs. Conclusions/Significance A peculiar epidemiological situation was observed in northeastern Italy, with the co-circulation of two distinct populations of L. infantum; one population mainly detected in dogs and the other population detected in humans and in a sand fly. While the classical cycle of CanL in Italy fits well into the data obtained for the first population, the population found in infected humans exhibits a different cycle, probably not involving a canine reservoir. This study can contribute to a better understanding of the population structure of L. infantum circulating in northeastern Italy, thus providing useful epidemiologic information for public health authorities., Author summary Visceral leishmaniasis is a sand fly-borne disease caused by protozoan parasites of the genus Leishmania. Leishmania infantum is the only parasitic species circulating in Italy and dogs are considered the main reservoirs of the disease. In this study, 55 L. infantum strains obtained from humans, dogs and sand flies from the Emiliana-Romagna (E-R) region, northeastern Italy, were assessed using multilocus microsatellite typing, a tool applied for population genetic studies. Results were compared with those obtained from 10 samples of visceral leishmaniasis cases occurring in other Italian regions and with 52 strains of the L. donovani complex from other foci of leishmaniasis. Our genetic analysis revealed that canine and human L. infantum strains from the E-R region were separated in two distinct populations; all samples obtained from dogs belonged to one population, while all but one human samples as well as a sand fly sample fell under another population. Samples from patients with visceral leishmaniasis from other Italian regions proved to be genetically similar to strains circulating in dogs. Our findings raise questions on the role of dogs as main reservoirs for human visceral leishmaniasis in the investigated area of northeastern Italy.

Migratory birds have an important role in transporting ticks and associated tick-borne pathogens over long distances. In this study, 2,793 migratory birds were captured by nets in a ringing station, located in northern Italy, and checked for the presence of ticks. Two-hundred and fifty-one ticks were identified as nymphs and larvae of Ixodes ricinus (Linnaeus, 1758) and they were PCR-screened for the presence of bacteria belonging to Borrelia burgdorferi sensu lato, Rickettsia spp., Francisella tularensis and Coxiella burnetii. Four species of Borrelia (B. garinii, B. afzelii, B. valaisiana and B. lusitaniae) and three species of Rickettsia (R. monacensis, R. helvetica and Candidatus Rickettsia mendelii) were detected in 74 (30%) and 25 (10%) respectively out of 251 ticks examined. Co-infection with Borrelia spp. and Rickettsia spp. in the same tick sample was encountered in 7 (7%) out of the 99 infected ticks. We report for the first time the presence of Candidatus Rickettsia mendelii in I. ricinus collected on birds in Italy. This study, besides confirming the role of birds in dispersal of I. ricinus, highlights an important route by which tick-borne pathogens might spread across different countries and from natural environments towards urbanised areas.

Usutu virus (USUV), a member of the genus Flavivirus, is known to circulate at low prevalence in Northern Italy, and has been reported to cause overt infection. USUV was first reported in Europe in 2001, but a retrospective study showed that it has been present in Italy at least since 1996. Seroprevalence data for USUV antibodies in sera are being collected in different European countries, showing circulation at low prevalence in human populations. Interestingly, two consecutive studies in Northern Italy indicate a possible increase in the presence of the virus, from 0% to 0.23% seroprevalence in blood donors. In this study, antibodies against USUV were measured in 3 consecutive blood samples collected from October 2014 to December 2015 from 33 forestry workers in the Po river valley, while samples from 200 blood donors from the same geographical area were tested in parallel. Neutralizing and IgG antibodies were found in six forestry workers (18.1%) and in two blood donors (1%). Our results indicate that USUV circulation in the examined area, part of a highly populated region in Northern Italy, is higher than expected. Healthy subjects exhibit a higher prevalence than what was found in a previous report in an adjoining region (0.23%), while the population at risk shows a much higher prevalence value (18.1%).

Salmonella enterica serovar Typhimurium (STm) and its monophasic variant 4,[5],12:i:- (VMSTm) have been responsible for an increased number of foodborne infections in humans in Europe in recent years. The aim of this study was to investigate the origin of three foodborne salmonellosis outbreaks that occurred in Pavia Province (Lombardy region, northern Italy) in 2010. Phenotypic and genetic characteristics of the STm and VMSTm isolates from patients and from food that were recovered in the framework of the three outbreaks were evaluated through serotyping, phage typing, antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE), and multiple-locus variable-number tandem repeat analysis (MLVA). Salami from three artisan producers, which had all purchased meat from the same slaughterhouse, was the food source of infection in outbreak I. STm isolates were recovered from salami and patients with symptoms of gastroenteritis. These isolates had the same PFGE type and the same rare MLVA profile (3-18-9-NA-211). The same molecular profiles were found in an STm isolate from a salami, which likely was the source of another family outbreak (II). A VMSTm strain with common phenotypic and molecular profiles was isolated from three hospitalized patients and identified as the cause of another putative outbreak (III). During the following 3 years (2011 through 2013), 360 salami produced in Pavia Province were monitored for the presence of S. enterica . In 2011, no STm and VMSTm isolates were recovered from 159 salami tested. During 2012 and 2013, 13.9% of 201 tested salami harbored S. enterica , and half of the isolates were VMSTm, mainly in salami from those artisan producers involved in the previous outbreaks. These isolates were genetically variable, especially in terms of MLVA profiles. The data collected suggest that from 2012, VMSTm has replaced STm in the environments of the salami producers monitored in this study, and these data confirm the dominance of this emergent serovar along the pork supply chain.

Background Two main genetic groups (B.12 and B.FTNF002-00) of Francisella tularensis ssp. holarctica are endemic in Europe. The B.FTNF002-00 group proved to be dominant in Western European countries, while strains of the B.12 group were isolated mainly in Northern, Central and Eastern Europe. The clinical course of tularemia in the European brown hare (Lepus europaeus) also shows distinct patterns according to the geographical area. Acute course of the disease is observed in hares in Western European countries, while signs of sub-acute or chronic infection are more frequently detected in the eastern part of the continent. The aim of the present study was to examine whether there is any difference in the virulence of the strains belonging to the B.FTNF002-00 and B.12 genetic clades. Results Experimental infection of Fischer 344 rats was performed by intra-peritoneal injection of three dilutions of a Hungarian (B.12 genotype) and an Italian (B.FTNF002-00 genotype) F. tularensis ssp. holarctica strain. Moderate difference was observed in the virulence of the two genotypes. Significant differences were observed in total weight loss values and scores of clinical signs between the two genotypes with more rats succumbing to tularemia in groups infected with the B.FTNF002-00 genotype. Conclusions Results of the experimental infection are consistent with previous clinical observations and pathological studies suggesting that F. tularensis ssp. holarctica genotype B.FTNF002-00 has higher pathogenic potential than the B.12 genotype. Electronic supplementary material The online version of this article (doi:10.1186/s12917-017-0968-9) contains supplementary material, which is available to authorized users.

Introduction Listeria monocytogenes (Lm) is a bacterium widely distributed in nature and able to contaminate food processing environments, including those of dairy products. Lm is a primary public health issue, due to the very low infectious dose and the ability to produce severe outcomes, in particular in elderly, newborns, pregnant women and immunocompromised patients. Methods In the period between April and July 2015, an increased number of cases of listeriosis was observed in the area of Pavia, Northern Italy. An epidemiological investigation identified a cheesemaking small organic farm as the possible origin of the outbreak. In this work we present the results of the retrospective epidemiological study that we performed using molecular biology and genomic epidemiology methods. The strains sampled from patients and those from the target farm's cheese were analyzed using PFGE and whole genome sequencing (WGS) based methods. The performed WGS based analyses included: a) in-silico MLST typing; b) SNPs calling and genetic distance evaluation; c) determination of the resistance and virulence genes profiles; d) SNPs based phylogenetic reconstruction. Results Three of the patient strains and all the cheese strains resulted to belong to the same phylogenetic cluster, in Sequence Type 29. A further accurate SNPs analysis revealed that two of the three patient strains and all the cheese strains were highly similar (0.8 SNPs of average distance) and exhibited a higer distance from the third patient isolate (9.4 SNPs of average distance). Discussion Despite the global agreement among the results of the PFGE and WGS epidemiological studies, the latter approach agree with epidemiological data in indicating that one the patient strains could have originated from a different source. This result highlights that WGS methods can allow to better.

Twelve Large White pigs were experimentally infected with 1000 Toxoplasma gondii oocysts/each. Serology was carried out at different time points post infection (p.i.) and animals were slaughtered at four months p.i. One of two thighs was examined for T. gondii infection status by PCR and bioassay in mice. The other thigh was processed for Parma ham production. Four thighs were examined after twelve months of curing, four after fourteen months and four were examined after sixteen months. Cured hams were analyzed by PCR, bioassay and in-vitro cultivation on Vero cells followed by real-time PCR. Pigs seroconverted from day 21 p.i. Bioassays were positive for all fresh thighs, but negative for cured hams. PCR was positive for parasite DNA from most thighs both at slaughter and post curing, but parasite growth was not observed following in vitro cultivation and real-time PCR. Results indicate that the curing process of Parma Ham (PDO), when carried out according to the Parma Ham consortium regulations, can inactivate T. gondii tissue cysts. Results would suggest that food-borne transmission of T. gondii to consumers from Parma ham can be excluded.

In this paper, we report an investigation on cat-scratch disease (CSD) in Northern Italy. Seventy-four cases of CSD were diagnosed at the San Matteo hospital, Pavia, during the period 2005-2010. Of these 74 patients, 18 (24.3 %) reported atypical clinical manifestations such as ocular papillitis, maculopapular eruptions, vertebral infection, pulmonary infiltrates, and granulomatous hepatitis. Contact with cats was documented for 61 patients (82.4 %), while cat-related trauma was reported for 49 patients (66.2 %). We subsequently investigated the presence of Bartonella infection in cats belonging to the above patients and in other domestic and stray cats from three provinces of Northern Italy. Among the 27 domestic cats tested, nine of the 11 belonging to the CSD patients and two of the remaining 16 were infected by B. henselae (81.8 % vs. 12.5 %). Out of over 1,300 stray cats examined, 23.1 % were seropositive for B. henselae; after culturing and genotyping, 17 % were found to be infected by B. henselae (15.5 %) or B. clarridgeiae (1.5 %).

Midichloria mitochondrii is an intracellular bacterium found in the hard tick Ixodes ricinus. In this arthropod, M. mitochondrii is observed in the oocytes and in other cells of the ovary, where the symbiont is present in the cell cytoplasm and inside the mitochondria. No studies have so far investigated whether M. mitochondrii is present in the salivary glands of the tick and whether it is transmitted to vertebrates during the tick blood meal. To address the above issues, we developed a recombinant antigen of M. mitochondrii (to screen human sera) and antibodies against this antigen (for the staining of the symbiont). Using these reagents we show that (i) M. mitochondrii is present in the salivary glands of I. ricinus and that (ii) seropositivity against M. mitochondrii is highly prevalent in humans parasitized by I. ricinus (58%), while it is very low in healthy individuals (1·2%). These results provide evidence that M. mitochondrii is released with the tick saliva and raise the possibility that M. mitochondrii is infectious to vertebrates. Besides this, our study indicates that M. mitochondrii should be regarded as a package of antigens inoculated into the human host during the tick bite. This implies that the immunology of the response toward the saliva of I. ricinus is to be reconsidered on the basis of potential effects of M. mitochondrii and poses the basis for the development of novel markers for investigating the exposure of humans and animals to this tick species.

Francisella tularensis subsp. holarctica isolates from Austria, Germany, Hungary, Italy, and Romania were placed into an existing phylogeographic framework. Isolates from Italy were assigned to phylogenetic group B.FTNF002-00; the other isolates, to group B.13. Most F. tularensis subsp. holarctica isolates from Europe belong to these 2 geographically segregated groups.

Recently, Italy-particularly the Emilia-Romagna region-was the location of consecutive outbreaks of human diseases caused by the arboviruses chikungunya virus and West Nile virus. The two outbreaks, spread by different species of mosquitoes, were not related, but pointed out the lack of an arboviral surveillance program in this region. Beginning in 2007 entomological surveillance was initiated in the Emilia-Romagna region, and in 2008 the program was improved and extended at Lombardia region. Using CO(2)-baited traps, 65,292 mosquitoes were collected; pooled by date of collection, location, and species; macerated manually; and tested by reverse transcription (RT)-polymerase chain reaction for the presence of alphaviruses, orthobunyaviruses, and flaviviruses. Amplicons were sequenced and employed for identification of viral RNA by basic local alignment search tool search in GenBank. Results of these assays showed (1) the presence of West Nile virus in two pools of Culex pipiens mosquitoes, (2) the presence of RNA of two orthobunyaviruses, Tahyna virus in a pool of Ochlerotatus caspius mosquitoes and Batai virus in a pool of Anopheles maculipennis mosquitoes, and (3) the presence of flavivirus RNAs in pools of Oc. caspius, Aedes albopictus, and Aedes vexans mosquitoes; the sequences of these amplicons were most closely related to flaviviruses that have been detected only in mosquitoes and had no recognized vertebrate host (Aedes flavivirus, Culex flavivirus, and Kamiti River virus).

Bartonella henselae is a fastidious bacterium associated with infections in humans and cats. The mechanisms involved in the long-term survival of bartonellae despite vigorous host immune responses are poorly understood. Generation of genetic variants is a possible strategy to circumvent the host specific immune responses. The authors have recently demonstrated the coexistence of different genetic variants within the progeny of three primary B. henselae isolates from Berlin by PFGE analysis. Aims of the present study were to determine whether coexistence of different variants is a common feature of B. henselae isolates worldwide and whether the genetic variants originally emerged in vivo. Thirty-four primary isolates from different geographical regions were analysed by subjecting multiple single-colony-derived cultures to PFGE analysis. Up to three genetic variants were detected within 20 (58.8 %) isolates, indicating that most primary isolates display a mosaic-like structure. The close relatedness of the genetic variants within an isolate was confirmed by multi-locus sequence typing. In contrast to the primary isolates, no genetic variants were detected within the progeny of 20 experimental clones generated in vitro from 20 primary isolates, suggesting that the variants were not induced in vitro during the procedure of PFGE analysis. Hence, the genetic variants within a primary isolate most likely originally emerged in vivo. Consideration of the mosaic structure of primary isolates is essential when interpreting typing studies on B. henselae.

BackgroundTularemia is a zoonosis caused by the Francisella tularensis, a highly infectious Gram-negative coccobacillus. Due to easy dissemination, multiple routes of infection, high environmental contamination and morbidity and mortality rates, Francisella is considered a potential bioterrorism threat and classified as a category A select agent by the CDC. Tick bites are among the most prevalent modes of transmission, and ticks have been indicated as a possible reservoir, although their reservoir competence has yet to be defined. Tick-borne transmission of F. tularensis was recognized in 1923, and transstadial transmission has been demonstrated in several tick species. Studies on transovarial transmission, however, have reported conflicting results.ObjectiveThe aim of this study was to evaluate the role of ticks as reservoirs for Francisella, assessing the transovarial transmission of F. tularensis subsp. holarctica in ticks, using experimentally-infected females of Dermacentor reticulatus and Ixodes ricinus.ResultsTransmission electron microscopy and fluorescence in situ hybridization showed F. tularensis within oocytes. However, cultures and bioassays of eggs and larvae were negative; in addition, microscopy techniques revealed bacterial degeneration/death in the oocytes.ConclusionsThese results suggest that bacterial death might occur in oocytes, preventing the transovarial transmission of Francisella. We can speculate that Francisella does not have a defined reservoir, but that rather various biological niches (e.g. ticks, rodents), that allow the bacterium to persist in the environment. Our results, suggesting that ticks are not competent for the bacterium vertical transmission, are congruent with this view.

Two healthy buffaloes (Bubalus bubalis) in a herd which had not been vaccinated against infectious bovine rhinotracheitis (IBR), were selected for their seropositivity for anti-bovine herpesvirus type 1 (BoHV-1) glycoprotein E antibodies, and injected intramuscularly daily with dexamethasone for five consecutive days (day 1 to day 5) to reactivate any latent herpesvirus. Blood samples and nasal and vaginal swabs were collected daily from day 5 to day 15 from each buffalo for virological examination. All the vaginal swabs and blood samples were negative, but 13 of the 22 nasal swabs were positive; a cytopathic effect was observed in primary cultures of bovine fetal lung cells, and the viral isolates were identified as a herpesvirus by PCR. The viral strains were characterised by the sequence analysis of the genes coding for glycoproteins D and B, and the gene sequences were then used for phylogenetic analysis. The isolates from both buffaloes appeared identical at the level of the two genes, and were more closely related to bovine herpesvirus type 5 than to BoHV-1.

A multiplex polymerase chain reaction (PCR) method coupled with a restriction analysis of PCR products (PCR with restriction fragment length polymorphism) was developed for the simultaneous detection of bovine herpesvirus 1, bovine herpesvirus 2, and bovine herpesvirus 4 infections. The specificity, sensitivity, and practical diagnostic applicability of this method were evaluated. This assay may be also adapted to the diagnosis of suid herpesvirus 1 and equine herpesviruses 1 and 3 and could become a powerful diagnostic tool.

Lyme disease, caused by Borrelia burgdorferi sensu lato (1), is endemic to various areas of Italy (2). The main vector of Lyme disease in Italy is the hard tick (Ixodes ricinus), a species widespread in mountain regions populated by wild ungulates (2). Residents of these areas and forestry workers are at risk for Lyme disease (3). Heavily populated flat regions are not considered as risk areas. For example, I. ricinus ticks have never been reported in the flat areas of the Po River Valley in the Lombardy region, one of the most important industrial districts in Europe and an area of intensive agriculture and livestock breeding. Human population density is high; >6 million persons reside in Milano and surrounding counties. In areas of Italy to which I. ricinus ticks are known to be endemic, physicians have appropriate awareness of the risks from tick bite and Lyme disease; outside these areas, awareness probably is not adequate. In late spring 2008, a forestry worker at a natural park west of Milano in the Po River Valley was treated for cutaneous mycosis on the basis of an erythematous rash on an arm. In August 2008, this patient described this skin alteration to one of us (C.B.). Subsequent clinical examination and serologic analyses led to diagnosis of Lyme disease. Because of this case, we investigated different areas of the park for ticks; collected ticks were screened by PCR for Lyme borreliae. We conducted a retrospective analysis of forestry workers in the area; 2 workers reporting the appearance of erythematous rash in the previous months underwent serologic analyses.

Coxiella burnetii , the aetiologic agent of Q fever (Query fever), is an intracellular Gram-negative bacterium belonging to the family of Coxiellaceae (Drancourt and Raoult 2005). Infections by C burnetii involve a wide range of susceptible hosts, ranging from mammals to arthropods (Angelakis and Raoult 2010). Abortion, stillbirth and non-viable neonates are the main clinical signs of infection in sheep and goats, while metritis, infertility and, rarely abortion, are typical for cattle (Arricau-Bouvery and Rodolakis 2005). C burnetii can produce spore-like forms, which are easily spread by wind and are able to survive for several months in the environment; for this reason the inhalation of contaminated aerosol and/or dust is considered the primary route of infection (Arricau-Bouvery and Rodolakis 2005). The consumption of contaminated raw milk does not seem to represent an efficient route of disease transmission (Rodolakis and others 2007), however, bulk tank milk (BTM) is an important specimen for epidemiological survey on dairy herds. In fact, even if C burnetii can be excreted in milk by infected animals continuously or intermittently (Guatteo and others 2007), milk is the most frequent way of shedding in asymptomatic cows (Rodolakis and others 2007). Many investigations have been performed among ruminant farms in European countries (Paiba and others 1999, Agger and others 2010, McCaughey …

a b s t r a c t Background: Specific bacterial infections or alterations of the gut microbiota likely trigger immuno- pathological phenomena associated with Crohn's disease and ulcerative colitis. Mycobacterium avium subspecies paratuberculosis is a candidate etiological agent of Crohn's disease. Definitive causal connec- tion between Mycobacterium avium subspecies paratuberculosis infection and Crohn's disease has not been demonstrated. Aims: To determine the circulation of Mycobacterium avium subspecies paratuberculosis in Crohn's disease patients and water supplies in an Italian region where this bacterium is endemic in cattle farms. Methods: Mycobacterium avium subspecies paratuberculosis screening was performed on biopsies from human patients, and from water samples, using two different PCR procedures. Results: In hospitals where multiple specimens were obtained from different sites in the intestine, the prevalence of Mycobacterium avium subspecies paratuberculosis infection was 82.1% and 40% respec- tively in Crohn's disease and ulcerative colitis patients; in another hospital, where single specimens were obtained from patients, the bacterium was not detected. Control subjects also harboured Mycobac- terium avium subspecies paratuberculosis, but at a lower prevalence. Tap water samples collected in the study area contained Mycobacterium avium subspecies paratuberculosis DNA. Discussion: The results of screenings for Mycobacterium avium subspecies paratuberculosis in humans are deeply influenced by both the number and location of the collected biopsies. There is a wide circulation of the organism in the study area, considering the prevalence in humans and its presence in drinking water.

Several zoonotic agents that infect wildlife may be transmitted to humans through contaminated game meat. Toxoplasma gondii and Trichinella spp. infect the muscular tissue at some stage of their biological cycle and consequently contaminate meat, while enteric bacteria (e.g. Salmonella spp., verocytotoxic Escherichia coli (VTEC), Yersinia enterocolitica) may contaminate the carcase after the animal’s death when the intestine is ruptured by shot pellets or during evisceration. Gutting and skinning of carcases may lead to contamination of humans with Mycobacterium bovis, Brucella spp., and Francisella tularensis, the etiological agents of bovine tuberculosis, brucellosis and tularaemia, respectively. We report here the occurrence of the above mentioned organisms over a 5-year period in some species of wild mammals of Lombardy and Emilia-Romagna, northern Italy. Trichinellae were recovered in 0.15% and 0.13% of samples from red foxes and wild boars, respectively. Antibodies to T. gondii were detected in a percentage ranging from 23.3% to 33.3% of wild ruminants and wild boars, and the parasite was also directly detected in European hares. Several serotypes of Salmonella spp. were recovered from cervids, red foxes and wild boars with prevalences from 1.46% to 18.7%. Faecal samples from roe deer tested negative for VTEC, while Y. enterocolitica was detected in about 8% samples from roe deer and from a few wild boars. M. bovis was rarely recovered from red deer and red foxes, and occasionally from wild boar, where M. microti, another species belonging to the Mycobacterium tuberculosis complex, occurred much more frequently. Antibodies to Brucella spp. were very rarely detected only in European hares, while F. tularensis was detected by PCR in about 7% European hares. This monitoring programme was conceived and implemented by the regional and local administrations in close collaboration with the official veterinary services and the hunters’ associations.

A 50-year-old man, pentathlete and fond of salmon fishing, after returning from Alaska was referred to our Division for an erythematous and indolent lesion on the right thigh gradually enlarging from the right knee to the right rear and the buttock.The patient was clinically diagnosed with figurate erythema. Laboratory analysis demonstrated a moderate leukocytosis and hypergammaglobulinemia, accompanied by serological markers of past contact with noted EBV and CMV.A “punch” biopsy of the edge of the lesion showed a superficial and deep perivascular and interstitial infiltrate of lymphocytes, eosinophiles and a few plasma cells, consistent with a borrelial infection. Lyme serology (IFI) suggested a questionable borderline positivity; subsequent investigations by ELISA and Western Blot were both positive, leading to the diagnosis of erythema chronicum migrans in course of borreliosis.The diagnosis was further confirmed by positive PCR for Borrelia burgdorferi sensu lato. The erythema resolved after a 2-week doxycycline treatment (2x100 mg die). The amplification product (16S rDNA gene) obtained from skin biopsy was sequenced using standard ABI technology, and confirmed the identification of a member of B. burgdorferi sensu lato (sl) complex. Since this sequence was not useful to identify the genospecies, further studies were conducted employing a nested PCR targeted on the internal transcribed spacer (ITS) of B. burgdorferi, following a protocol previously described (1). The ITS sequence showed 100% identity with B. afzelii, a species not reported from North America, thus prompting us to conclude that the patient was not infected by B. afzelii during the fishing trip in Alaska.

Class II major histocompatibility complex (MHCII) is required for the presentation of antigens to CD4 helper T cells. During nephritis, not only primary antigen presenting cells such as histiocytes and lymphocytes, but also cytokine-stimulated tubular epithelial cells express MHCII. Leptospirosis in fattening pigs is characterized by several degrees of nephritis, from absence of lesions to severe multifocal tubulo-interstitial inflammation. Renal tissue from 20 8-month-old pigs with spontaneous nephritis and 6 control pigs without renal lesions were investigated for leptospirosis by indirect immunohistochemistry (IHC) and polymerase chain reaction (PCR). IHC for MHCII also was performed on renal samples. Serum samples were tested for different serovars of Leptospira interrogans. Control pigs were free of interstitial nephritis and negative for leptospirosis by all tests. In pigs with nephritis, serology was positive for serovar Pomona in 19/20 pigs. In 16 of these 19 pigs, leptospiral renal infection was confirmed by PCR and/or indirect IHC. Nephritic lesions were classified histologically into perivascular lymphocytic (4 pigs), lymphofollicular (6 pigs), lymphohistiocytic (8 pigs), and neutrophilic (2 pigs) pattern. MHCII expression by histiocytes and lymphocytes was observed in all lesions. Prominent MHCII expression in regenerating tubular epithelium was observed in lymphofollicular and lymphohistiocytic nephritis. No tubular colocalization between leptospiral and MHCII antigen was observed. Results suggest that during leptospiral nephritis, MHCII contributes to the intensity of the inflammatory response. Furthermore de novo MHCII expression in regenerating tubules may play a role in the defence mechanism against leptospiral tubular colonization.

Seventy-two pigs were examined for the presence of leptospires in the kidney by both bacteriological culture and an immunoperoxidase procedure performed on formalin-fixed, paraffin-embedded sections of tissue with a primary antibody raised in rabbits against serovar pomona. The methods were in accordance in 62 of 70 (89 per cent) of the specimens. Compared with culture the sensitivity of the immunoperoxidase procedure was 30 of 38 (78 per cent) and its specificity 100 per cent; the predictive value of a positive result was 100 per cent, of a negative result, 80 per cent. The major advantages of the immunoperoxidase procedure are specificity, speed of execution and the possibility of simultaneous visualisation of leptospiral antigen and microscopic lesion.

Poly-γ-glutamate (γ-PGA) is an industrially interesting polymer secreted mainly by members of the class Bacilli which forms a shield able to protect bacteria from phagocytosis and phages. Few enzymes are known to degrade γ-PGA; among them is a phage-encoded γ-PGA hydrolase, PghP. The supposed role of PghP in phages is to ensure access to the surface of bacterial cells by dismantling the γ-PGA barrier. We identified four unannotated B. subtilis genes through similarity of their encoded products to PghP; in fact these genes reside in prophage elements of B. subtilis genome. The recombinant products of two of them demonstrate efficient polymer degradation, confirming that sequence similarity reflects functional homology. Genes encoding similar γ-PGA hydrolases were identified in phages specific for the order Bacillales and in numerous microbial genomes, not only belonging to that order. The distribution of the γ-PGA biosynthesis operon was also investigated with a bioinformatics approach; it was found that the list of organisms endowed with γ-PGA biosynthetic functions is larger than expected and includes several pathogenic species. Moreover in non-Bacillales bacteria the predicted γ-PGA hydrolase genes are preferentially found in species that do not have the genetic asset for polymer production. Our findings suggest that γ-PGA hydrolase genes might have spread across microbial genomes via horizontal exchanges rather than via phage infection. We hypothesize that, in natural habitats rich in γ-PGA supplied by producer organisms, the availability of hydrolases that release glutamate oligomers from γ-PGA might be a beneficial trait under positive selection.

Bartonella henselae is the major etiological agent of cat scratch disease in humans. Cats act as the natural reservoir of B. henselae and can transmit the infection to humans by a bite or scratch. The prevalence of B. henselae in cat populations was evaluated by serological and bacteriological tests. A total of 769 stray cats from three urban and three rural areas in northern Italy were sampled between January 1999 and December 2000. The positive and the negative predictive values of serological tests with respect to bacteremic status were evaluated. Tests of a total of 140 cats (18%) resulted in detection of bacteremia. A total of 540 cats were tested by serology; 207 (38%) were seropositive. Of the 531 cats tested by both methods, the results for 65 (12.2%) showed both bacteremia detection and seropositivity. The molecular typing of the isolates showed that 20.6% of bacteremic cats were infected with B. henselae type I strain, 61.1% were infected with B. henselae type II, and 18.3% were coinfected with both. A statistically significant difference in antibody and bacteremia prevalences among geographical areas was detected. Statistical analysis showed no association between characteristics such as seroprevalence-bacteremic status, sex, general health status, and the presence of ectoparasites. The negative predictive value of serological test was 84.7%, and the positive predictive value was 31.8%. Receiving operator characteristic analysis of the data showed that serological tests had a low predictive value in relation to the bacteremic status of a cat; in surveys aimed at assessing the real risk of B. henselae infection in a human population, therefore, we suggest the use of blood culture as the reference test. Nevertheless, both blood culture assays and serological tests for Bartonella infection should be performed for a complete evaluation of the health status of cats.

Bartonella henselae has been identified and characterized for the first time in Italy. A strain, designed Pavia-1, was isolated from the blood of a cat whose owner developed cat scratch disease (CSD). Pavia-1 and two American B. henselae strains (Houston-1, ATCC 49882, type I and strain 269608, UC Davis, type II) were compared by whole-cell fatty analysis (CFA), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for protein profiles, Western immunoblotting (WB) for reactivity with polyclonal antibodies, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), type-specific 16S rRNA PCRs, and pulsed-field gel electrophoresis (PFGE). Bartonella clarridgeiae (ATCC 51734) was also included for comparison. Pavia-1 was identified as a B. henselae type I. PFGE allowed differentiation between B. clarridgeiae and B. henselae and furthermore, between all the B. henselae strains. The fingerprints of PFGE observed for Pavia-1 were distinct from those of B. henselae type II and also of Houston-1, suggesting that the two type I strains derived from two different clones. These results show the capability of B. henselae to develop genotypic variability between genetically related strains.

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.

The authors describe an outbreak of Pacheco's Parrot Disease (PPD) which occurred in Italy in recently imported psittacine birds and was caused by Psittacid Herpesvirus type 2 (PsiHV2). The authors stress the different susceptibility to the disease in the species involved. This outbreak showed the failure of the vaccine prophylaxis that had been administered to the birds with ordinary commercial preparations containing Psittacid Herpesvirus type 1. The authors emphasize the necessity of producing a vaccine containing inactivated viruses of all known serotypes.

In order to clarify the supposed involvement of urban pigeons (Columba livia livia) in the epidemiology of Lyme disease, the presence of antibodies against Borrelia burgdorferi and Borrelia anserina in pigeons' sera, collected in 12 areas of northern and central Italy, was evaluated. This evaluation was carried out using a classic immunofluorescence assay (IFA), a surface immunofluorescence assay (SIFA) and a standard Western Blot (WB) assay. A total of 104 out of 3,186 (3.26%) serum samples were positive for both spirochetes when tested by IFA, with titres ranging from 1/40 to 1/1280. All positive specimens showed the same or a higher reactivity against B. anserina than against B. burgdorferi. Of the IFA positive samples, 30 were tested by WB and SIFA to evaluate further the specificity of the antibody response, i.e. to try to clarify against which spirochete the antibodies were raised. The presence of antibodies against the 23 kDa protein exclusive to B. anserina, and against epitopes which are not surface-exposed and which are common to B. anserina and B. burgdorferi, was assessed by WB and SIFA. No serological evidence that B. burgdorferi can infect pigeons was found.

An outer surface lipoprotein of 22 kDa was identified in the avian pathogen Borrelia anserina Ni-NL by using antibody preparations reactive with bacterial surface-exposed proteins. Amino acid sequence analysis of the 22-kDa protein demonstrated 90% identity with VmpA of B. turicatae , suggesting that the protein belongs to the family of 20-kDa outer surface proteins of the genus Borrelia . All of the 60 chicks intramuscularly treated with antibodies specifically reacting with the 22-kDa protein and infected with strain Ni-NL were completely protected from infection, since no spirochetemia was detected, and from death. Control chicks were treated with immune sera raised against apathogenic strain B. anserina Es, which expresses a prominent 20-kDa polypeptide that is also a member of the Vmp family but does not cross-react immunologically with the 22-kDa protein of the Ni-NL strain. These animals, infected with B. anserina Ni-NL, showed a high degree of spirochetemia 10 days after infection, and all died between 14 and 21 days after infection. The results showed that the 22-kDa surface protein of B. anserina Ni-NL is a determinant of the pathogenic potential of the strain and also confirmed that only strain-specific antibodies are protective against B. anserina infection.