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Amanita phalloides, Lepiota cristata, Lepiota brunneoincarnata and Inocybe asterospora are among the most important species responsible of mushroom poisoning in northern Italy. A real time PCR method for the identification of samples containing DNA from each of these species was developed. To test specificity all protocols were applied on DNA extracted from various mushroom species; sensitivity was assessed performing serial dilutions on all samples; versatility of the protocols was evaluated performing tests on DNA extracted from different matrices. The protocols showed high sensitivity (32 ng dried mushroom), high specificity and sensitive detection of DNA extracted from difficult samples, including pasta with mushroom, cooked mushrooms and gastric aspirates.

Wolbachia pipientis is possibly the most widespread endosymbiont of arthropods and nematodes. While all Wolbachia strains have historically been defined as a single species, 16 monophyletic clusters of diversity (called supergroups) have been described. Different supergroups have distinct host ranges and symbiotic relationships, ranging from mutualism to reproductive manipulation. In filarial nematodes, which include parasites responsible for major diseases of humans (such as Onchocerca volvulus , agent of river blindness) and companion animals ( Dirofilaria immitis, the dog heartworm), Wolbachia has an obligate mutualist role and is the target of new treatment regimens. Here, we compare the genomes of eight Wolbachia strains, spanning the diversity of the major supergroups (A–F), analysing synteny, transposable element content, GC skew and gene loss or gain. We detected genomic features that differ between Wolbachia supergroups, most notably in the C and D clades from filarial nematodes. In particular, strains from supergroup C (symbionts of O. volvulus and D. immitis ) present a pattern of GC skew, conserved synteny and lack of transposable elements, unique in the Wolbachia genus. These features could be the consequence of a distinct symbiotic relationship between C Wolbachia strains and their hosts, highlighting underappreciated differences between the mutualistic supergroups found within filarial nematodes.

Multidrug-resistant (MDR) Klebsiella pneumoniae is one of the most important causes of nosocomial infections worldwide. After the spread of strains resistant to beta-lactams at the end of the previous century, the diffusion of isolates resistant to carbapenems and colistin is now reducing treatment options and the containment of infections. Carbapenem-resistant K. pneumoniae strains have spread rapidly among Italian hospitals, with four subclades of pandemic clonal group 258 (CG258). Here we show that a single Italian hospital has been invaded by three of these subclades within 27 months, thus replicating on a small scale the “Italian scenario.” We identified a single clone responsible for an epidemic outbreak involving seven patients, and we reconstructed its star-like pattern of diffusion within the intensive care unit. This epidemiological picture was obtained through phylogenomic analysis of 16 carbapenem-resistant K. pneumoniae isolates collected in the hospital during a 27-month period, which were added to a database of 319 genomes representing the available global diversity of K. pneumoniae strains. Phenotypic and molecular assays did not reveal virulence or resistance determinants specific for the outbreak isolates. Other factors, rather than selective advantages, might have caused the outbreak. Finally, analyses allowed us to identify a major subclade of CG258 composed of strains bearing the yersiniabactin virulence factor. Our work demonstrates how the use of combined phenotypic, molecular, and whole-genome sequencing techniques can help to identify quickly and to characterize accurately the spread of MDR pathogens.

We announce the draft genome sequence of Clostridium tyrobutyricum strain DIVETGP. This strain was isolated from cow's milk used for Grana Padano cheese production. The genome was obtained using Illumina HiSeq technology and comprises 45 contigs for 3,018,999 bp, with a G+C content of 30.8%.

International audience; Hard ticks are hematophagous arthropods that act as vectors of numerous pathogenic microorganisms of high relevance in human and veterinary medicine. Ixodes ricinus is one of the most important tick species in Europe, due to its role of vector of pathogenic bacteria such as Borrelia burgdorferi and Anaplasma phagocytophilum, of viruses such as tick borne encephalitis virus and of protozoans as Babesia spp. In addition to these pathogens, I. ricinus harbors a symbiotic bacterium, Midichloria mitochondrii. This is the dominant bacteria associated to I. ricinus, but its biological role is not yet understood. Most M. mitochondrii symbionts are localized in the tick ovaries, and they are transmitted to the progeny. M. mitochondrii bacteria have however also been detected in the salivary glands and saliva of I. ricinus, as well as in the blood of vertebrate hosts of the tick, prompting the hypothesis of an infectious role of this bacterium. To investigate, from a proteomic point of view, the tick I. ricinus and its symbiont, we generated the protein profile of the ovary tissue (OT) and of salivary glands (SG) of adult females of this tick species. To compare the OT and SG profiles, 2-DE profiling followed by LC-MS/MS protein identification were performed. We detected 21 spots showing significant differences in the relative abundance between the OT and SG, ten of which showed 4- to 18-fold increase/decrease in density. This work allowed to establish a method to characterize the proteome of I. ricinus, and to detect multiple proteins that exhibit a differential expression profile in OT and SG. Additionally, we were able to use an immunoproteomic approach to detect a protein from the symbiont. Finally, the method here developed will pave the way for future studies on the proteomics of I. ricinus, with the goals of better understanding the biology of this vector and of its symbiont M. mitochondrii

International audience; Midichloria mitochondrii is the most prevalent symbiont of the hard tick Ixodes ricinus, present in 100% of eggs and adult females of wild ticks. This bacterium is intracellular, and is the only known symbiont able to invade the mitochondria of the host cells. However, the role that M. mitochondrii plays in the host metabolism has yet to be elucidated. Multiple lines of evidence indicate the possibility of transmission of this bacterium to the vertebrate host during the tick blood meal. In order to investigate the role of M. mitochondrii in the biology of the tick host, we performed an antibiotic treatment on Ixodes ricinus individuals, with the aim of reducing/eliminating the symbiont, and to potentially observe the dynamic of bacterial infection in the tick host. We microinjected engorged adult females of I. ricinus with tetracycline, and we allowed the resulting larvae to feed on gerbils treated with the same antibiotic. The amount of M. mitochondrii was evaluated at different stages of the experiment using molecular techniques. In addition we evaluated the presence/absence of the symbiont DNA in the blood of gerbils used for the larval feeding. The performed treatments did not allow to eliminate the symbiont population from the host tick, however it allowed to reduce the multiplication that occurs after the larval blood meal. These results open the way for future experiments, using different antibiotic molecules, different administration methods and antibiotic administration on subsequent tick stages, to fulfill the goal of eliminating M. mitochondrii from the host I. ricinus, a major step in our understanding of the impact of this bacterium on ticks

Keywords: Midichloriaceae family, Oncorhynchusmykiss, real-time PCR, red mark syndrome.Red mark syndrome (RMS) is a chronic skin dis-ease of unknown aetiology affecting farmed rain-bow trout Oncorhynchus mykiss (Walbaum) inEurope. It consists of single or multiple skinlesions usually localized on the trunk of fishapproaching market size. RMS was first reportedin Scotland in 2003 but has subsequently beenobserved in other European countries, includingItaly (Verner-Jeffreys et al. 2008; Schmidt-Post-haus et al. 2009). This disease is generally associ-ated with water temperatures below 16 °C andcan still be present at 2 °C (Verner-Jeffreys et al.2008; McCarthy et al. 2013). RMS causes brightred lesions that tend to heal spontaneously, withno effects on weight or behaviour and the healingtime seems to be associated with the water tem-perature (Ferguson et al. 2006). Histologically,severe lymphohistiocytic dermatitis is reportedwith scale resorption (Ferguson et al. 2006; Nogu-era 2008; Verner-Jeffreys et al. 2008; Schmidt-Posthaus et al. 2010). In some RMS-affected indi-viduals, acute necrotizing myocarditis,inflammation of intestinal muscle, splenic conges-tion, peritonitis and perivascular lymphocyte infil-trate can be observed (Oidtmann et al. 2013).Although RMS is not lethal, its morbidity canreach up to 60% in fish culture and can thuscause important economic losses, due to down-grading of the product (Schmidt-Posthaus et al.2009; Oidtmann et al. 2013). In the UnitedStates, a similar condition of O. mykiss is knownas strawberry disease (SD) and the skin pathologyassociated with it resembles that of RMS.No aetiological agent has been unequivocallyidentified for RMS or for SD, although differentauthors suggest that a single transmissible agentcould be responsible for both (Lloyd et al. 2011).Oman (1990) reported experimental infectionthrough inoculation with SD lesion homogenate(Lloyd et al. 2008). Ferguson et al. (2006) sug-gested a possible association with Flavobacteriumpsychrophilum, but no data confirmed this hypoth-esis. Experimental cohabitation between fishaffected by RMS and na€ive ones demonstrated thetransmissibility of this pathology (Verner-Jeffreyset al. 2008). Moreover, histological analysisshowed acute inflammation in the area of the skinlesion with the presence of neutrophils and pro-tein oedema (Metselaar et al. 2010), suggesting abacterial infection.Lloyd et al. (2008) presented PCR evidence forthe presence of a Rickettsia-like organism (RLO)

Sawflies are important pests of various plant species. Diprion pini (L.) and Neodiprion sertifer (Geoffroy) (Hymenoptera: Diprionidae) are two of the most important sawfly pests in Italy, and both species are parasitized by the hymenopteran parasitoid Dahlbominus fuscipennis (Zetterstedt). Bacterial endosymbionts are currently studied for their high potential in strategies of biocontrol in a number of insect species. In this study, we investigated the presence of symbiotic bacteria ( Wolbachia and Cardinium ) in the three species of hymenoptera mentioned earlier, both in wild and laboratory populations. Although all samples were negative for the presence of Cardinium , 100% prevalence for Wolbachia was detected, as all examined individuals resulted to be PCR positive. Furthermore, 16S rDNA and ftsZ gene sequencing indicated that all individuals from the three hymenopteran species are infected by a single Wolbachia strain. Additionally, we report the presence of gynandromorphic individuals in D. pini , both in wild and laboratory-reared populations. Heat treatments on D. pini colonies removed the Wolbachia symbionts, but they also prevented the development of adults.

The aim of this study was to evaluate the antibacterial activity and cytotoxic effect of the compound SIAB-GV3, a new formulation presenting as an aqueous suspension of silicon dioxide (SiO2) functionalized with silver (Ag+) nanoparticles and benzalkonium. The product is formulated as an adjuvant for the treatment of inflammatory diseases of the oral cavity. This study demonstrates that SIAB-GV3 possesses strong antimicrobial activity against most of the common oral pathogens, in particular against Streptococcus pyogenes, Porphyromonas gingivalis and Aggregatibacter actinimycetemcomitans. The cytotoxic effect against human embryo lung fibroblasts (HELF cells) was evaluated at different times, from 1 h to 168 h, using concentrations of SIAB-GV3 ranging from 50 mg/ml to 0.0008 mg/ml. At the concentration of 10 mg/ml, commonly used in clinical practice, the compound results cyto- toxic after about 2 hours, this time being much longer than the typical time of local application, which is no more than 10 minutes. In conclusion, our findings suggest that SIAB-GV3 has a good antibacterial activity against the most common oral pathogens even at very low concentrations and a low cytotoxic activity, thanks to the synergistic effects of Ag nanoparticles, silicon dioxide and benzalkonium.

Group B Streptococcus (GBS) is considered to be the major cause of neonatal sepsis and meningitis of bacterial origin. Late-onset GBS infection is infrequent and occurs between 1 week and 3 months of age. The transmission of GBS through the ingestion of breast milk is reported in the literature, but only a few of these cases have been confirmed by molecular techniques. In this article we report five cases of late-onset GBS disease: transmission through maternal milk was confirmed in four cases, using the random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) typing assay. In addition, the RAPD-PCR assay showed that each of the isolated clones belonged to a different RAPD genotype, thus revealing that the late-onset GBS infections were not epidemiologically related.

The ancestors of mitochondria, or proto-mitochondria, played a crucial role in the evolution of eukaryotic cells and derived from symbiotic α-proteobacteria which merged with other microorganisms - the basis of the widely accepted endosymbiotic theory. However, the identity and relatives of proto-mitochondria remain elusive. Here we show that methylotrophic α-proteobacteria could be the closest living models for mitochondrial ancestors. We reached this conclusion after reconstructing the possible evolutionary pathways of the bioenergy systems of proto-mitochondria with a genomic survey of extant α-proteobacteria. Results obtained with complementary molecular and genetic analyses of diverse bioenergetic proteins converge in indicating the pathway stemming from methylotrophic bacteria as the most probable route of mitochondrial evolution. Contrary to other α-proteobacteria, methylotrophs show transition forms for the bioenergetic systems analysed. Our approach of focusing on these bioenergetic systems overcomes the phylogenetic impasse that has previously complicated the search for mitochondrial ancestors. Moreover, our results provide a new perspective for experimentally re-evolving mitochondria from extant bacteria and in the future produce synthetic mitochondria.

Background Proteins from the ABC family (ATP-binding cassette) represent the largest known group of efflux pumps, responsible for transporting specific molecules across lipid membranes in both prokaryotic and eukaryotic organisms. In arthropods they have been shown to play a role in insecticide defense/resistance. The presence of ABC transporters and their possible association with insecticide transport have not yet been investigated in the mosquito Anopheles stephensi, the major vector of human malaria in the Middle East and South Asian regions. Here we investigated the presence and role of ABCs in transport of permethrin insecticide in a susceptible strain of this mosquito species. Methods To identify ABC transporter genes we obtained a transcriptome from untreated larvae of An. stephensi and then compared it with the annotated transcriptome of Anopheles gambiae. To analyse the association between ABC transporters and permethrin we conducted bioassays with permethrin alone and in combination with an ABC inhibitor, and then we investigated expression profiles of the identified genes in larvae exposed to permethrin. Results Bioassays showed an increased mortality of mosquitoes when permethrin was used in combination with the ABC-transporter inhibitor. Genes for ABC transporters were detected in the transcriptome, and five were selected (AnstABCB2, AnstABCB3, AnstABCB4, AnstABCmember6 and AnstABCG4). An increased expression in one of them (AnstABCG4) was observed in larvae exposed to the LD50 dose of permethrin. Contrary to what was found in other insect species, no up-regulation was observed in the AnstABCB genes. Conclusions Our results show for the first time the involvement of ABC transporters in larval defense against permethrin in An. stephensi and, more in general, confirm the role of ABC transporters in insecticide defense. The differences observed with previous studies highlight the need of further research as, despite the growing number of studies on ABC transporters in insects, the heterogeneity of the results available at present does not allow us to infer general trends in ABC transporter-insecticide interactions. Electronic supplementary material The online version of this article (doi:10.1186/1756-3305-7-349) contains supplementary material, which is available to authorized users.

In insects, ABC transporters have been shown to contribute to defence/resistance to insecticides by reducing toxic concentrations in cells/tissues. Despite the extensive studies about this detoxifying mechanism, the temporal patterns of ABC transporter activation have been poorly investigated. Using the malaria vector Anopheles stephensi as a study system, we investigated the expression profile of ABC genes belonging to different subfamilies in permethrin-treated larvae at different time points (30 min to 48 h). Our results showed that the expression of ABCB and ABCG subfamily genes was upregulated at 1 h after treatment, with the highest expression observed at 6 h. Therefore, future investigations on the temporal dynamics of ABC gene expression will allow a better implementation of insecticide treatment regimens, including the use of specific inhibitors of ABC efflux pumps.

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.

Background The Midichloriaceae is a novel family of the order Rickettsiales, that encompasses intracellular bacteria associated with hard ticks (Ixodidae) and other arthropods. The most intensively investigated member of this family is Midichloria mitochondrii, a symbiotic bacterium of the sheep tick Ixodes ricinus, characterized by the capacity of multiplying inside the mitochondria. A recent study suggested that these bacteria might be inoculated into the human host during the tick bite. The purpose of this study was to determine the potential infectivity of Midichloria bacteria for non-human animals exposed to the risk of tick bite. Methods Blood from horses, cattle, sheep and dogs exposed to the risk of tick bite was included in this study. DNAs were extracted, and amplified using 16S ribosomal RNA primers conserved in the Midichloria genus. Furthermore, sera from dogs exposed to the risk of tick bite were analyzed in order to evaluate the presence of antibodies against the recombinant flagellar protein (rFliD) from M. mitochondrii using an ELISA test. Results Here we present two lines of evidence that support the possibility that bacteria from the genus Midichloria are inoculated into vertebrate hosts during a tick bite: (i) a direct evidence, i.e. the detection of circulating DNA from bacteria related with M. mitochondrii, in the blood of vertebrates exposed to tick parasitism; (ii) a further indirect evidence, i.e. the presence of antibodies against an antigen from M. mitochondrii in dogs exposed to the risk of tick bite. It is interesting to note that variability was detected in the Midichloria gene sequences recovered from positive animals, and that some of these sequences were identical to those generated from tick-associated Midichloria. Conclusions Based on the results, and on the overall information so far published on the genus Midichloria, we suggest that these bacteria are likely to represent a novel group of vector-borne agents, with the potential of infecting mammalian hosts. Whether inoculation of Midichloria bacteria could cause a true infection and pathological alteration in mammalian hosts is still to be determined. Surely, results emphasize the relevance of Midichloria bacteria in investigations on tick immunology and tick-bite markers.

We announce the draft genome sequence of Salmonella enterica subsp. enterica serovar Manhattan strain 111113, isolated from a patient during an outbreak in northern Italy. The genome, which was obtained with Illumina MiSeq technology, is composed of 21 contigs for a total of 4,684,342 bp, with a G+C content of 52.17%.

We report the draft genome sequence of the Stenotrophomonas maltophilia strain EPM1, found in association with a culture of Giardia duodenalis . The draft genome sequence of S. maltophilia strain EPM1, obtained with Roche 454 GS-FLX Titanium technology, is composed of 19 contigs totaling 4,785,869 bp, with a G+C content of 66.37%.

Sequence type 258 (ST258) is the most widespread multidrug resistant (MDR) Klebsiella pneumoniae strain worldwide. Here, we report the draft genome sequences of two colistin-resistant MDR K. pneumoniae ST258 clinical strains isolated from hospital patients in Italy. These strains are resistant to β-lactams, cephalosporins, fluoroquinolones, aminoglycosides, macrolides, tetracyclines, carbapenems, and colistin.

“ Candidatus Midichloria mitochondrii” is an intramitochondrial bacterium of the order Rickettsiales associated with the sheep tick Ixodes ricinus . Bacteria phylogenetically related to “ Ca . Midichloria mitochondrii” (midichloria and like organisms [MALOs]) have been shown to be associated with a wide range of hosts, from amoebae to a variety of animals, including humans. Despite numerous studies focused on specific members of the MALO group, no comprehensive phylogenetic and statistical analyses have so far been performed on the group as a whole. Here, we present a multidisciplinary investigation based on 16S rRNA gene sequences using both phylogenetic and statistical methods, thereby analyzing MALOs in the overall framework of the Rickettsiales . This study revealed that (i) MALOs form a monophyletic group; (ii) the MALO group is structured into distinct subgroups, verifying current genera as significant evolutionary units and identifying several subclades that could represent novel genera; (iii) the MALO group ranks at the level of described Rickettsiales families, leading to the proposal of the novel family “ Candidatus Midichloriaceae.” In addition, based on the phylogenetic trees generated, we present an evolutionary scenario to interpret the distribution and life history transitions of these microorganisms associated with highly divergent eukaryotic hosts: we suggest that aquatic/environmental protista have acted as evolutionary reservoirs for members of this novel family, from which one or more lineages with the capacity of infecting metazoa have evolved.

Here, we present an investigation on the spatial distribution of the bacterial symbiont Candidatus Midichloria mitochondrii within Ixodes ricinus, by whole mount fluorescence in situ hybridization (FISH). M. mitochondrii is a peculiar, recently discovered bacterium that resides in the mitochondria of female ticks. We applied a rapid and specific FISH protocol with oligonucleotide probes targeted on the 16S rRNA of M. mitochondrii, 12S rRNA of tick mitochondria, and a probe revealing active mitochondria. In this report that represents the first application of whole mount FISH on ticks, we observed strong, specific fluorescence signals in all the examined life stages, as the optimized protocol allowed us to overcome the autofluorescence interference of the cuticle. Cellular localization and quantification of the symbionts were also assessed with electron microscopy and specific real-time PCR, respectively.

Molecular methods are important tools in the diagnosis of bloodstream bacterial infections, in particular in patients treated with antimicrobial therapy, due to their quick turn-around time. Here we describe a new broad-range real-time PCR targeting the 23S rDNA gene and capable to detect as low as 10 plasmid copies per reaction of targeted bacterial 23S rDNA gene. Two commercially available DNA extraction kits were evaluated to assess their efficiency for the extraction of plasma and whole blood samples spiked with different amount of eitherStaphylococcus aureusorEscherichia coli, in order to find the optimal extraction method to be used. Manual QIAmp extraction method with enzyme pre-treatment resulted the most sensitive for detection of bacterial load. Sensitivity of this novel assay ranged between 10 and 103 CFU per PCR reaction forE. coliandS. aureusin human whole blood samples depending on the extraction methods used. Analysis of plasma samples showed a 10- to 100-fold reduction of bacterial 23S rDNA in comparison to the corresponding whole blood specimens, thus indicating that whole blood is the preferential sample type to be used in this real-time PCR protocol. Our results thus show that the 23S rDNA gene represents an optimal target for bacteria quantification in human whole blood.

Here, we present the draft genome sequence of Klebsiella pneumoniae subsp. pneumoniae sequence type 512 (ST512) isolated during a KPC-producer outbreak. This strain is resistant to β-lactams, cephalosporins, fluoroquinolones, aminoglycosides, macrolides, tetracyclines, and carbapenems but susceptible to colistin. The ST512-K30BO genome is composed of 289 contigs for 5,392,844 bp with 56.9% G+C content.

In the last few years, improved analytical tools and the integration of genetic data with multiple sources of information have shown that temperate species exhibited more complex responses to ice ages than previously thought. In this study, we investigated how Pleistocene climatic changes affected the current distribution and genetic diversity of European populations of the tick Ixodes ricinus, an ectoparasite with high ecological plasticity. We first used mitochondrial and nuclear genetic markers to investigate the phylogeographic structure of the species and its Pleistocene history using coalescent-based methods; then we used species distribution modelling to infer the climatic niche of the species at last glacial maximum; finally, we reviewed the literature on the I. ricinus hosts to identify the locations of their glacial refugia. Our results support the scenario that during the last glacial phase, I. ricinus never experienced a prolonged allopatric divergence in separate glacial refugia, but persisted with interconnected populations across Southern and Central Europe. The generalist behaviour in host choice of I. ricinus would have played a major role in maintaining connections between its populations. Although most of the hosts persisted in separate refugia, from the point of view of I. ricinus, they represented a continuity of 'bridges' among populations. Our study highlights the importance of species-specific ecology in affecting responses to Pleistocene glacial-interglacial cycles. Together with other cases in Europe and elsewhere, it contributes to setting new hypotheses on how species with wide ecological plasticity coped with Pleistocene climatic changes.

In modern taxonomy, DNA barcoding is particularly useful where biometric parameters are difficult to determine or useless owing to the poor quality of samples. These situations are frequent in parasitology. Here, we present an integrated study, based on both DNA barcoding and morphological analysis, on cestodes belonging to the genus Taenia, for which biodiversity is still largely underestimated. In particular, we characterized cestodes from Italian wildcats (Felis silvestris silvestris), free-ranging domestic cats (Felis silvestris catus) and hybrids populations. Adult taeniids were collected by post-mortem examinations of the hosts and morphologically identified as Taenia taeniaeformis. We produced cox1 barcode sequences for all the analysed specimens, and we compared them with reference sequences of individuals belonging to the genus Taenia retrieved from GenBank. In order to evaluate the performance of a DNA barcoding approach to discriminate these parasites, the strength of correlation between species identification based on classical morphology and the molecular divergence of cox1 sequences was measured. Our study provides clear evidence that DNA barcoding is highly efficient to reveal the presence of cryptic lineages within already-described taeniid species. Indeed, we detected three well-defined molecular lineages within the whole panel of specimens morphologically identified as T. taeniaeformis. Two of these molecular groups were already identified by other authors and should be ranked at species level. The third molecular group encompasses only samples collected in Italy during this study, and it represents a third candidate species, still morphologically undescribed.

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.

According to Bergey’s Manual of Systematic Bacteriology, the Rickettsiales are ‘…bacteria with typical Gram-negative cell walls and no flagella’. The recently sequenced genome of ‘Candidatus Midichloria mitochondrii’, a divergent lineage within the order Rickettsiales capable of invading mitochondria in ixodid ticks, revealed the presence of 26 putative flagellar genes. Open questions in relation to this observation are whether these genes are expressed and whether they possess the domains expected for the flagellar function. Here we show that: (a) the putative flagellar proteins of ‘Ca. M. mitochondrii’ actually possess the conserved domains and structural features required for their function in a model bacterium; (b) the seven flagellar genes of ‘Ca. M. mitochondrii’ that have been tested are expressed at the RNA level; and (c) the putative flagellar cap gene of this bacterium (FliD) is expressed at the protein level, and can be stained within the bacterium and at its surface. Beside the specific questions that we have addressed that relate to the first evidence, to our knowledge, for a flagellar apparatus in a member of the order Rickettsiales, we present here novel tools (recombinant protein and antibodies) that will facilitate the study of ‘Ca. M. mitochondrii’.

Bovine viral diarrhea virus (BVDV) is a widespread and economically important pathogen of cattle; genetic typing of BVDV isolates distinguished two species, namely BVDV-1 and BVDV-2. BVDV-1 is the most widespread worldwide and it includes at least 11 subtypes. With the aim of clarifying the routes of circulation of BVDV-1 subtypes in an endemic area and in order to investigate the relationships between the genetic diversity of BVDV and its geographic distribution, a phylogenetic analysis of 5′ untranslated region of Italian sequences was performed using a new Bayesian framework allowing the spatial–temporal reconstruction of the evolutionary dynamics of highly variable viruses. Our analyses suggested that different BVDV subtypes entered the North-Eastern part of Italy at different times within a time span between 23 and 7 years ago. The largest virus dispersion occurred between the mid 1990s and the early 2000s. A possible gravity-like dynamic of the infection, originating in larger animal population then following patterns of national commercial-flow, should be hypothesized.

The initiation of the intracellular symbiosis that would give rise to mitochondria and eukaryotes was a major event in the history of life on earth. Hypotheses to explain eukaryogenesis fall into two broad and competing categories: those proposing that the host was a phagocytotic proto-eukaryote that preyed upon the free-living mitochondrial ancestor (hereafter FMA), and those proposing that the host was an archaebacterium that engaged in syntrophy with the FMA. Of key importance to these hypotheses are whether the FMA was motile or nonmotile, and the atmospheric conditions under which the FMA thrived. Reconstructions of the FMA based on genome content of Rickettsiales representatives-generally considered to be the closest living relatives of mitochondria-indicate that it was nonmotile and aerobic. We have sequenced the genome of Candidatus Midichloria mitochondrii, a novel and phylogenetically divergent member of the Rickettsiales. We found that it possesses unique gene sets found in no other Rickettsiales, including 26 genes associated with flagellar assembly, and a cbb(3)-type cytochrome oxidase. Phylogenomic analyses show that these genes were inherited in a vertical fashion from an ancestral alpha-proteobacterium, and indicate that the FMA possessed a flagellum, and could undergo oxidative phosphorylation under both aerobic and microoxic conditions. These results indicate that the FMA played a more active and potentially parasitic role in eukaryogenesis than currently appreciated and provide an explanation for how the symbiosis could have evolved under low levels of oxygen.

Lactoferrin (Lf) is a non-haem iron-binding glycoprotein with a molecular weight of about 80 kDa, synthesized by glandular epithelial cells and stored in the secondary granules of neutrophils. The physiological significance of Lf is related to non-specific immune defence against pathogens, immunomodulatory activity, iron homeostasis, antioxidant properties and regulation of cell growth. Lf is a bioactive component of the mammary secretions and its modulatory and defensive functions do affect the newborn and the mammary gland as well. In this work a bovine mammary epithelial cell line (BME-UV1) was used as an in vitro model of the bovine mammary epithelium to examine the protective role of exogenous bovine Lf (bLf) against the cytotoxic damage induced by bacterial lipopolysaccharides (LPS) and the endogenous bLf mRNA expression after LPS exposure. In the in vitro model used, exogenous bLf exerts a protective effect against endotoxin cytotoxicity, which could be mediated by the LPS-neutralizing capability of bLf. In addition, in BME-UV1 cells the response to LPS exposure does not involve bLf mRNA expression, suggesting that this cell line lack of functional LPS-responsive elements.

Intracellular bacteria are widespread in nature and may adopt a wide array of life styles. They can be found free in the cytoplasm of their host cells, within host-derived vacuoles, or even in the nucleus. Here, we review current knowledge about ‘Candidatus Midichloria mitochondrii’, an intracellular bacterium that invades the host mitochondria in the ixodid tick Ixodes ricinus. This bacterium was first detected by electron microscopy in two independent studies, published in, 1979 and, 1992, that showed it in the cells of the ovary in adult ticks and also in the cells of the ovarian primordia in larvae and nymphs. This symbiont resides not only in the cytoplasm, but also inside the mitochondria of the ovarian cells, where it appears to penetrate the outer mitochondrial membrane and colonize the intermembrane space. Molecular studies have recently been performed, addressing the phylogenetic position, transmission, and prevalence of this novel bacterium. The functional significance of this symbiotic association has yet to be revealed. Even though ‘Candidatus M. mitochondrii’ seems to behave as a ‘predator’ towards the host mitochondria, this does not appear to interfere with egg development, thus ensuring the vertical transmission of the bacteria to the progeny. The 100% prevalence in the ovaries of females of I. ricinus may indicate a mutualistic association, whereas the peculiar intramitochondrial localization suggests that ‘Candidatus M. mitochondrii’ might be exploiting the energy available in the mitochondrial environment. The possibility that the bacterium is a reproductive parasite should also be considered.

“ Candidatus Midichloria mitochondrii,” an intracellular symbiont of the tick Ixodes ricinus , is the only described organism able to invade the mitochondria of any multicellular organism. We used quantitative PCR to examine cycles of bacterial growth and death throughout the host's development and found that they correspond with the phases of engorgement and molt, respectively.

The causative agents of avian mycobacteriosis in pet birds are rarely identified. The aim of this study is to add information about the etiology of avian mycobacteriosis. The identification of mycobacterium species in 27 cases of avian mycobacteriosis in pet birds was investigated by polymerase chain reaction (PCR) and sequencing of a rRNA hypervariable region. Avian mycobacteriosis appeared to be an infrequent diagnosis. Interestingly, a few cases of avian mycobacteriosis were recorded in very young birds. The most commonly affected species were the canary (Serinus canarius), the Eurasian goldfinch (Carduelis carduelis) and the red siskin (Spinus cucullatus). All but one bird were infected with Mycobacterium genavense. Mycobacterium avium was identified only in one case.

SUMMARYThe hard tickIxodes ricinus(Ixodidae) is the sole animal thus far shown to harbour an intra-mitochondrial bacterium, which has recently been namedMidichloria mitochondrii. The objectives of this work were (i) to screen ixodid ticks forMidichloria-related bacteria and (ii) to determine whether these bacteria exploit the intra-mitochondrial niche in other tick species. Our main goal was to discover further models of this peculiar form of symbiosis. We have thus performed a PCR screening forMidichloria-related bacteria in samples of ixodid ticks collected in Italy, North America and Iceland. A total of 7 newly examined species from 5 genera were found positive for bacteria closely related toM. mitochondrii. Samples of the tick speciesRhipicephalus bursa, found positive in the PCR screening, were analysed with transmission electron microscopy, which revealed the presence of bacteria both in the cytoplasm and in the mitochondria of the oocytes. There is thus evidence that bacteria invade mitochondria in at least 2 tick species. Phylogenetic analysis on the bacterial 16S rRNA gene sequences generated from positive specimens revealed that the bacteria form a monophyletic group within the order Rickettsiales. The phylogeny ofMidichloriasymbionts and related bacteria does not appear completely congruent with the phylogeny of the hosts.

We thank Lavinia Simoncini for assistance with cloning and DNA quantification and Claudio Genchi for discussion. N.L. was supported by the Australian Research Council; International audience; The tick Ixodes ricinus is responsible for the transmission of a number of bacterial, protozoan and viral diseases to humans and animals in Europe and Northern Africa. Female I. ricinus from England, Switzerland and Italy have been found to harbour an intracellular alpha-proteobacterium, designated IricES1, within the cells of the ovary. IricES1 is the only prokaryote known to exist within the mitochondria of any animal or multicellular organism. To further examine the distribution, prevalence and mode of transmission of IricES1, we performed polymerase chain reaction screening of I. ricinus adults from 12 countries across its geographic distribution, including tick colonies that have been maintained in the laboratory for varying periods of time. IricES1 was detected in 100% of field-collected female ticks from all countries examined (n = 128), while 44% of males were found to be infected (n = 108). Those males that are infected appear to harbour fewer bacteria than females. Sequencing of fragments of the 16S rRNA and gyrB genes revealed very low nucleotide diversity among various populations of IricES1. Transmission of IricES1 from engorged adult females to eggs was found to be 100% (n = 31). In tick colonies that had been maintained in the laboratory for several years, a relatively low prevalence was found in females (32%; n = 25). To our knowledge, IricES1 is the most widespread and highly prevalent of any tick-associated symbiont.

Babesia caballi and Theileria equi are the causative agents of equine piroplasmosis. In this epidemiological study, 294 horses reared in a rural area of northern Italy were studied. During January 2008–January 2009, blood samples were taken for serology (indirect fluorescent antibody test) and for polymerase chain reaction (PCR). Data on the geographical area, sex, and age were collected for statistical analysis of risk factors associated with infection. A seroprevalence of 8.5% was found: 8.2% of the animals were positive for anti–T. equi antibodies and 0.3% for anti–B. caballi antibodies. No dual infections were observed. Of those horses with positive serology to T. equi, 33% were also positive in PCR, whereas none of the seropositive horses for B. caballi was positive in PCR. No significant correlation between sex or age was found for infection status.

According to the tRNA punctuation model, the mitochondrial genome (mtDNA) of mammals and arthropods is transcribed as large polycistronic precursors that are maturated by endonucleolytic cleavage at tRNA borders and RNA polyadenylation. Starting from the newly sequenced mtDNA of Ixodes ricinus and using a combination of mitogenomics and transcriptional analyses, we found that in all currently-sequenced tick lineages (Prostriata, Metastriata and Argasidae) the 3'-end of the polyadenylated nad1 and rrnL transcripts does not follow the tRNA punctuation model and is located upstream of a degenerate 17-bp DNA motif. A slightly different motif is also present downstream the 3'-end of nad1 transcripts in the primitive chelicerate Limulus polyphemus and in Drosophila species, indicating the ancient origin and the evolutionary conservation of this motif in arthropods. The transcriptional analyses suggest that this motif directs the 3'-end formation of the nad1/rrnL mature RNAs, likely working as a transcription termination signal or a processing signal of precursor transcripts. Moreover, as most regulatory elements, this motif is characterized by a taxon-specific evolution. Although this signal is not exclusive of ticks, making a play on words it has been named "Tick-Box", since it is a check mark that has to be verified for the 3'-end formation of some mt transcripts, and its consensus sequence has been here carefully characterized in ticks. Indeed, in the whole mtDNA of all ticks, the Tick-Box is always present downstream of nad1 and rrnL, mainly in non-coding regions (NCRs) and occasionally within trnL(CUN). However, some metastriates present a third Tick-Box at an intriguing site - inside the small NCR located at one end of a 3.4 kb translocated region, the other end of which exhibits the nad1 Tick-Box - hinting that this motif could have been involved in metastriate gene order rearrangements.

Members of the bacterial order Rickettsiales are obligatorily associated with a wide range of eukaryotic hosts. Their evolutionary trajectories, in particular concerning the origin of shared or differential traits among distant sub-lineages, are still poorly understood. Here we characterised a novel Rickettsiales bacterium associated with the ciliate Paramecium tredecaurelia, and phylogenetically related to the Rickettsia genus. Its genome encodes significant lineage-specific features, chiefly the mevalonate pathway gene repertoire, involved in isoprenoid precursor biosynthesis. Not only this pathway has never been described in Rickettsiales, it also is very rare among bacteria, though typical in eukaryotes, thus likely representing a horizontally-acquired trait. The presence of these genes could enable an efficient exploitation of host-derived intermediates for isoprenoid synthesis. Moreover, we hypothesise the reversed reactions could have replaced canonical pathways for producing acetyl-CoA, essential for phospholipid biosynthesis. Additionally, we detected phylogenetically unrelated mevalonate pathway genes in metagenome-derived Rickettsiales sequences, likely indicating evolutionary convergent effects of independent horizontal gene transfer events. Accordingly, convergence, involving both gene acquisitions and losses, is highlighted as a relevant evolutionary phenomenon in Rickettsiales, possibly favoured by plasticity and comparable lifestyles, representing a potentially hidden origin of other more nuanced similarities among sub-lineages. This article is protected by copyright. All rights reserved.

Klebsiella oxytocacauses opportunistic human infections and post-antibiotic haemorrhagic diarrhoea. ThisEnterobacteriaceaespecies is genetically heterogeneous and is currently subdivided into seven phylogroups (Ko1 to Ko4, Ko6 to Ko8). Here we investigated the taxonomic status of phylogroups Ko3 and Ko4. Genomic sequence-based phylogenetic analyses demonstrate that Ko3 and Ko4 formed well-defined sequence clusters related to, but distinct from,Klebsiella michiganensis(Ko1),Klebsiella oxytoca(Ko2),K. huaxiensis(Ko8) andK. grimontii(Ko6). The average nucleotide identity of Ko3 and Ko4 were 90.7% withK. huaxiensisand 95.5% withK. grimontii, respectively. In addition, three strains ofK. huaxiensis, a species so far described based on a single strain from a urinary tract infection patient in China, were isolated from cattle and human faeces. Biochemical and MALDI-ToF mass spectrometry analysis allowed differentiating Ko3, Ko4 and Ko8 from the otherK. oxytocaspecies. Based on these results, we propose the namesKlebsiella spallanzaniifor the Ko3 phylogroup, with SPARK_775_C1T(CIP 111695T, DSM 109531T) as type strain, andKlebsiella pasteuriifor Ko4, with SPARK_836_C1T(CIP 111696T, DSM 109530T) as type strain. Strains ofK. spallanzaniiwere isolated from human urine, cow faeces and farm surfaces, while strains ofK. pasteuriiwere found in faecal carriage from humans, cows and turtles.Accession numbersThe nucleotide sequences generated in this study were deposited in ENA and are available through the INSDC databases under accession numbersMN091365(SB6411T= SPARK775C1T), MN091366 (SB6412T= SPARK836C1T) and MN104661 to MN104677 (16S rRNA), MN076606 to MN076643 (gyrAandrpoB), and MN030558 to MN030567 (blaOXY). Complete genomic sequences were submitted to European Nucleotide Archive under the BioProject number PRJEB15325.AbbreviationsANI, average nucleotide identity; HCCA, a-cyano-4-hydroxycinnamic acid; isDDH,in silicoDNA-DNA hybridization; SCAI, Simmons citrate agar with inositol; MALDI57 ToF MS: Matrix-assisted laser desorption/ionization time of flight mass spectrometry

Taxonomy is the science of defining and naming groups of biological organisms based on shared characteristics and, more recently, on evolutionary relationships. With the birth of novel genomics/bioinformatics techniques and the increasing interest in microbiome studies, a further advance of taxonomic discipline appears not only possible but highly desirable. The present work proposes a new approach to modern taxonomy, consisting in the inclusion of novel descriptors in the organism characterization: (1) the presence of associated microorganisms (e.g.: symbionts, microbiome), (2) the mitochondrial genome of the host, (3) the symbiont genome. This approach aims to provide a deeper comprehension of the evolutionary/ecological dimensions of organisms since their very first description. Particularly interesting, are those complexes formed by the host plus associated microorganisms, that in the present study we refer to as “holobionts”. We illustrate this approach through the description of the ciliate Euplotes vanleeuwenhoeki sp. nov. and its bacterial endosymbiont “Candidatus Pinguicoccus supinus” gen. nov., sp. nov. The endosymbiont possesses an extremely reduced genome (~ 163 kbp); intriguingly, this suggests a high integration between host and symbiont.