In light of these findings, one could argue that the observation of an object we are used to manipulating modulates the corticospinal excitability of parts of the primary motor cortex that control muscles implicated in this action. Here we recorded EMG activity from the FDI muscle, which is at least partially involved in grasping objects of the size and shape of a mobile phone. The interesting finding we observed in this respect is that corticospinal excitability was modulated by the ownership of the seen object, in that it was larger following the presentation of an owned,

as compared with a non-owned mobile phone. While this result may suggest a specific functional organization of the motor cortex for our objects, this conclusion is partially at odd with studies that Venetoclax concentration analysed the difference of motor excitability during the observation of graspable vs. non-graspable objects (e.g. Buccino et al., 2005), or the time course of changes in motor excitability before the execution of grasping movements, as compared GSI-IX with the mere observation of an object (Prabhu et al., 2007). Overall, activation due to graspability processes emerges within a short time-window after the presentation of a graspable object. Buccino

et al. (2005), for example, found a difference between graspable and non-graspable objects 200 ms after object presentation. Prabhu et al. (2007) reported that corticospinal excitability

was augmented only when it was measured about 100 ms before the actual grasping execution, whereas no changes were manifest during passive observation of a graspable object (i.e. outside the mental set of performing an action). In light of these reports, and the absence of any change in corticospinal excitability observed here when stimulating the left hemisphere, we can dismiss the hypothesis that the increase in excitability of the right hemisphere observed when subjects were displayed either Self or Other mobile phones could be ascribed to general effects of graspability. Finally, it appeared that corporeal (hand) and non-corporeal stimuli (phone) contributed to the increase in corticospinal excitability observed at later time intervals (600 and 900 ms), provided many that they belonged to the observer (Self condition). Besides extending our knowledge of self-processes to hand and hand-associated objects, the present findings also provide insight about the time course of these processes, by showing that consistent MEP increase can be observed at relatively late timings. Previous studies focusing on hand stimuli did not explore the time course of self-hand processing (Patuzzo et al., 2003; Funase et al., 2007). In contrast, the temporal profile of self-related processing has been investigated in studies using face stimuli. Théoret et al.

Our analysis indicates the presence of a ‘core keratitis cluster’, associated with corneal infections, that is related to the P. aeruginosa eccB clonal complex, which is associated with adaptation to survival in environmental

water. This suggests that adaptation to environmental water is a key factor in the ability of P. aeruginosa to cause eye infections. Bacterial infection of the cornea (keratitis) is a serious ocular disease associated with significant visual loss Epigenetic inhibition and visually disabling scarring in 22–40% of cases, despite treatment with antimicrobials (Cheng et al., 1999; Schaefer et al., 2001; Bourcier et al., 2003). Visual loss is strongly associated with keratitis caused by Gram-negative bacteria rather than by Gram-positive bacteria (Keay et al., 2006).The incidence of bacterial keratitis is sixfold higher in contact lens wearers compared to the general population (Lam et al., 2002; Bourcier et al., 2003), and in contact lens wearers, Pseudomonas aeruginosa is the most common species isolated (Dutta et al., 2012; Stapleton & Carnt, 2012). In a UK study, 23% of 772 isolates collected from patients with bacterial keratitis were P. aeruginosa (Sueke et al., 2010), a pathogen associated with larger ulcers and worse outcomes compared

selleck kinase inhibitor to other bacteria causing keratitis (Kaye et al., 2010). A number of P. aeruginosa virulence factors have been implicated in keratitis, including elastase B, twitching motility associated with type IV pili, flagella, type III-secretion system (TTSS) and proteases, including protease IV (O’Callaghan et al., 1996; Fleiszig et al., 1997; Winstanley et al., 2005; Zhu et al., 2006; Choy et al., 2008). P. aeruginosa strains can be sub-divided into either cytotoxic (associated with ExoU) or invasive

(associated with ExoS), with cytotoxic Rucaparib molecular weight strains being significantly diminished in their invasive capability in vitro (Fleiszig et al., 1996; Feltman et al., 2001). Various studies have addressed the role of TTSS exoproducts in association with ocular infections (Fleiszig et al., 1996, 1997; Lomholt et al., 2001; Lee et al., 2003; Tam et al., 2007). These studies revealed that exoU-positive strains are associated with greater morbidity in P. aeruginosa infection (Finck-Barbancon et al., 1997). Moreover, isolates from keratitis are disproportionately carriers of exoU (rather than exoS) in comparison with the wider P. aeruginosa population (Winstanley et al., 2005). Since 2003, the University of Liverpool has served as a repository for bacterial isolates from patients with keratitis from six UK centres: London, Birmingham, Bristol, Newcastle, Manchester and Liverpool. These centres comprise the Microbiology Ophthalmic Group (MOG). In previous studies, we analysed 63 P. aeruginosa isolates collected between 2003 and 2004 from patients with keratitis (Winstanley et al., 2005; Stewart et al., 2011).

Motor performance was assessed by

a blinded rater using: non-dominant handwriting time and legibility, and mentally trained task at baseline (pre) and immediately after (post) mental practice combined with tDCS. Active tDCS significantly enhances the motor-imagery-induced improvement in motor function as compared with sham tDCS. There was a specific effect for the site of stimulation such that effects were only observed after M1 and DLPFC stimulation during mental practice. These findings provide new insights into motor imagery training and point out that two cortical targets (M1 and DLPFC) selleck inhibitor are significantly associated with the neuroplastic effects of mental imagery on motor learning. Further studies should explore a similar paradigm in patients with brain lesions. Mental practice (MP) is a training method in which a specific action is cognitively repeated without inducing learn more any actual movement for the intention of acquiring motor skill and enhancing motor performance (Grouios, 1992). Several studies have shown that MP improves motor skill performance in healthy people and in different patient populations (for a review, see Dickstein & Deutsch, 2007). For instance, in individuals who are healthy, these improvements of performance include gains in muscular force (Ranganathan et al., 2004) and upper limb

kinematics (Gentili et al., 2006). In the field of neurological rehabilitation, for example, promising findings have been reported for enhancing sit-to-stand performance and activities of daily living in people after stroke (Liu et al., 2004; Malouin et al., 2004; Page et al., 2005). Although it is clear that MP enhances physical performance, the neural mechanisms underlying this effect are unknown. It has been proposed SSR128129E that imagined movement shares similar neural substrates with those that are involved in executed motor actions (Decety, 1996a,b; Guillot et al., 2008).

Indeed, as shown by neuroimaging studies, imagined actions are associated with functional and structural changes in a wide range of neural structures including the premotor and supplementary motor area (SMA) (Ingvar & Philipson, 1977; Roland et al., 1980; Decety et al., 1990, 1994), primary motor cortex (M1) (Porro et al., 1996; Ehrsson et al., 2003; Kuhtz-Buschbeck et al., 2003; Solodkin et al., 2004), cerebellum and basal ganglia (Decety et al., 1994; Lafleur et al., 2002; Naito et al., 2002; Guillot et al., 2008). The dorsolateral prefrontal cortex of the left hemisphere seems also to be involved in imagined movement (Decety et al., 1994). Despite evidence of engagement of these cerebral substrates during motor imagery, the specific role of each area in the MP effects on motor learning have not been clarified.

4 Clark MA, Hartley buy Ribociclib A, Geh JI. Cancer of the anal canal. Lancet Oncol 2004; 5: 149–157. 5 Kim JH, Sarani B, Orkin BA et al. HIV-positive patients with anal carcinoma have poorer treatment tolerance and outcome than HIV-negative patients. Dis Colon Rectum 2001; 44: 1496–1502. 6 Chiao EY, Giordano TP, Richardson P, El-Serag HB. Human immunodeficiency virus-associated squamous cell cancer of the anus: epidemiology and outcomes in the highly active antiretroviral therapy era. J Clin Oncol 2008; 26: 474–479. 7 Shiels MS, Pfeiffer RM, Engels EA. Age at cancer diagnosis among persons with AIDS in the

United States. Ann Intern Med 2010; 153: 452–460. 8 Kreuter A, Potthoff A, Brockmeyer NH et al. Anal carcinoma in human immunodeficiency virus-positive men: results of a prospective study from Germany. Br J Dermatol 2010; 162: 1269–1277. 9 Piketty C, Selinger-Leneman H, Bouvier AM et al. Incidence of HIV-related selleck products anal cancer remains increased despite long-term combined antiretroviral treatment: results from the French Hospital Database on HIV. J Clin Oncol 2012; 30: 4360–4366. 10 Silverberg MJ, Lau B, Justice AC et al. Risk

of anal cancer in HIV-infected and HIV-uninfected individuals in North America. Clin Infect Dis 2012; 54: 1026–1034. 11 Bower M, Powles T, Newsom-Davis T et al. HIV-associated anal cancer: has highly active antiretroviral therapy reduced the incidence or improved the outcome? J Acquir Immune Defic Syndr 2004; 37: 1563–1565. 12 Clifford GM, Polesel

J, Rickenbach M et al. Cancer risk in the Swiss HIV Cohort Study: associations with immunodeficiency, smoking, and highly active antiretroviral therapy. J Natl Cancer Inst 2005; 97: 425–432. 13 Diamond C, Taylor TH, Aboumrad T et al. Increased Phosphoribosylglycinamide formyltransferase incidence of squamous cell anal cancer among men with AIDS in the era of highly active antiretroviral therapy. Sex Transm Dis 2005; 32: 314–320. 14 Engels EA, Pfeiffer RM, Goedert JJ et al. Trends in cancer risk among people with AIDS in the United States 1980–2002. AIDS 2006; 20: 1645–1654. 15 Piketty C, Selinger-Leneman H, Grabar S et al. Marked increase in the incidence of invasive anal cancer among HIV-infected patients despite treatment with combination antiretroviral therapy. AIDS 2008; 22: 1203–1211. 16 Franceschi S, Lise M, Clifford GM et al. Changing patterns of cancer incidence in the early- and late-HAART periods: the Swiss HIV Cohort Study. Br J Cancer 2010; 103: 416–422. 17 Crum-Cianflone NF, Hullsiek KH, Marconi VC et al. Anal cancers among HIV-infected persons: HAART is not slowing rising incidence. AIDS 2010; 24: 535–543. 18 Beckmann AM, Daling JR, Sherman KJ et al. Human papillomavirus infection and anal cancer. Int J Cancer 1989; 43: 1042–1049. 19 Palefsky JM. Anal squamous intraepithelial lesions: relation to HIV and human papillomavirus infection. J Acquir Immune Defic Syndr 1999; 21(Suppl 1): S42–48. 20 Machalek DA, Poynten M, Jin F et al.

PCR assays were also performed using the genomic DNA of 47 pathogenic and 33 reference strains of S. suis serotypes (types

1–31, 33 and 1/2) as a template to test the distribution of the HP0272 gene with the following pair of primers: forward primer, 5′-GTTGGATCCGAATCGCTAGAAC-3′; reverse primer, 5′-TATCTCGAGACTTGCTTCGCCTGTAT-3′. Data were analysed using Student’s t-test; a value of P<0.05 was considered to indicate statistically significant differences. As shown in Fig. 1a, the purified recombinant HP0272 showed a protein band of approximately 130 kDa upon sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE). Although the learn more apparent sizes were greater than the theoretical sizes, the identity of purified recombinant HP0272 could be confirmed by MS. An analysis of the predicted HP0272 amino acid sequence revealed CP-868596 an LPNTG consensus motif typical of membrane-anchored surface proteins of many gram-positive bacteria at the C terminus and a putative signal-peptidase cleavage site between Ala44 and Glu45 (Fig. 1b). Two repeats of an

88 amino-acid sequence with a 28 amino-acid sequence overlap were detected within the carboxyl half of the protein (Fig. 1c). Furthermore, a conserved domain blast search identified a lipoprotein domain in the Thr500–Met655 region, which exhibited 36% similarity to the outer membrane lipoprotein A from Actinobacillus pleuropneumoniae. To monitor the antigen-specific response provided by immunization with recombinant HP0272, humoral-mediated responses were evaluated in immunized mice. Antibody titres against recombinant HP0272 were determined in sera obtained from mice on the 10th day after the booster injection. Levels of specific IgG titres against recombinant HP0272 were significantly higher in the immunized group (P<0.001) than in the Thiamet G negative control group (Fig. 2a). To reveal the type of immune response, the IgG1 and IgG2a subclasses were determined as surrogate markers to indicate T helper 1 (Th1) responses (IgG2a antibodies) and Th2 responses (IgG1 antibodies). Although the nature of these experiments did

not allow accurate quantification of different immunoglobulin subclasses, they did indicate that IgG1 responses predominated over IgG2a responses (Fig. 2b). Ten days after the booster immunization, all mice were challenged by with a lethal dose of 2 × 109 CFU of log-phase SS2 ZYS. In the four groups (Fig. 3), all of the mice in the blank group (group 4) and 62.5% in the negative control group (group 3) died, whereas 100% of mice from the recombinant HP0272 (group 1) and the positive control group (group 2) survived on day 1. The remaining three mice in the negative control group exhibited significant clinical signs (e.g. ruffled hair coat, slow response to stimuli), while mice in the recombinant HP0272 immunized group and the positive control group showed weaker clinical signs.

There is the potential for interprofessional education to increase appropriate utilisation of pathology services to improve antibiotic prescribing in this group of patients. Anna Murphy1,2, Larry Goodyear2, Peter Rivers2, Cheng Xie3, Anjali Shah2, Mayuri Parmer2 1University Hospitals of Leicester NHS Trust, Leicester, UK, 2DeMontfort University, Leicester, UK, 3The First Affiliated Hospital of Suzhou University, Nanjing, China An accurate assessment of inhaler technique is essential but reliable evaluation may be difficult to achieve find protocol due

to the subjectivity of the observer. Lowest agreement between observers was seen in the coordination of actuation and inhalation technique steps Inter-educator agreement for inhaler evaluation is difficult to obtain with certain steps being more difficult Inhaled medicines are the cornerstone of therapy in obstructive lung disease. Correct inhaler technique is essential to achieve optimal therapeutic BYL719 response. A large proportion of people prescribed inhalers do not use them correctly.1 Checklist-based

assessment and correction of step-by-step technique is an effective strategy for improving inhaler technique.1 However, reliable evaluation of inhaler technique may be difficult to achieve due to the subjectivity of the educator. A pilot study was designed to investigate the error rate for the different inhaler technique steps and to examine the level of agreement between two observers of inhaler demonstrations. Twenty-four patients selected opportunistically had their

inhaler technique assessed using metered dose inhalers (MDIs) against the 7-step inhaler technique checklist devised at University Hospitals of Leicester. Each patient was asked to demonstrate their technique to a respiratory pharmacist and a research pharmacist – both previously trained on inhaler technique assessment. The pharmacists separately scored each step as correct/incorrect/unsure. If any step was incorrect in the opinion of the respiratory pharmacist the patient was counselled and the observation repeated. click here Using the same method, 12 patients were assessed with each of MDI plus aerochamber and turbohaler and 10 with the accuhaler device. Appropriate NHS and University ethics opinions and approvals were obtained Overall, observation revealed that none of the 24 patients achieved correct technique for all steps. On first demonstration both observers noted correct technique for only 12 (50%) patients for the key steps of breathing-out and holding breath after inhalation. Only 2 patients (8%) were observed as having the correct inspiration rate for optimal drug deposition. This was improved to 84.6% when the MDI was combined with an aerochamber. Twenty patients were assessed a second time for the technique and based on all observations (n = 44) for the key stages Kappa scores ranged from 0.

, 2004). Here, we

present a newly devised PCR-RFLP method to differentiate M.tb from other members of the MTC on the basis of the −6T/C narK2 promoter SNP. Because none of these PCR-RFLP methods can fully differentiate the MTC members, a combination of different PCR-RFLP assays can improve the confidence of diagnosis. Previously, the −215T/C SNP upstream of narGHJI was correlated with differential nitrate reduction in MTC (Stermann et al., 2004); M. tb having the −215T genotype could reduce nitrate, whereas other members having the −215C genotype could not reduce nitrate. However, an extension of this analysis to more strains refuted this hypothesis on the basis of high nitrate reductase activity in M. canetti (−215C genotype) and some other ancestral strains of the M. tb−215C genotype (Goh et al., 2005). Interestingly, the nitrate reductase activity of MTC strains correlates better with the −6T/C SNP present in the narK2X promoter. VX-809 mw Both M. tb and M. canetti have the −6T genotype and both can reduce nitrate, whereas M. africanum, M. microti, M. bovis and BCG have the −6C genotype and all lack nitrate reductase activity. Because the −6C genotype results

in completely abolishing narK2X promoter activity, it is likely that all these species lack functional narK2X genes and this promoter defect is one of the key factors contributing to the lack of inducible nitrate reductase activity in them. The plasmid pNarG-GM1 was a generous gift provided by Dr C.D. Sohaskey. We sincerely acknowledge Dr H.K. Prasad, India, for providing genomic

DNA of clinical isolates of the MTC. This work was financially supported by a grant to J.S.T. check details from the Department of Biotechnology, Government of India. S.C. is grateful to CSIR for a research associateship. We acknowledge the facilities of the Biotechnology Information Systems (BTIS), Department of Biotechnology, and Government of India. “
“Methanococcus maripaludis has two surface appendages, namely flagella and pili. Flagella have been shown to be required for swimming, but no specific role has been assigned as yet to pili. In this report, wild-type M. maripaludis cells are compared with mutants lacking either pili or flagella or both surface appendages Ribonucleotide reductase in their ability to attach to a variety of surfaces including nickel, gold and molybdenum grids as well as glass, silicon and mica. Wild-type cells attached to varying degrees to all surfaces tested, except mica, via their flagella as observed by scanning electron microscopy. Large cables of flagella were found to leave the cell and to be unwound on the surface. In addition, such cables were often found to connect cells. In contrast, cells lacking either flagella or pili or both surface appendages were unable to attach efficiently to any surfaces. This indicates a second role for flagella in addition to swimming in M. maripaludis, as well as a first role for pili in this organism, namely in surface attachment.

In another classical conditioning MEG study with two different CS tones, Kluge et al. (2011) reported stronger tangential field gradients at left and right central sensor clusters at mid-latency (85–115 ms) and late (180–270 ms) intervals for CS+ (with omitted electric shock) compared to CS− tones during conditioning. When comparing all CS+ (also CS+ with US presentation) with CS− tones they found relatively enhanced gradient

fields for CS− processing in these sensor groups in an early interval between 30 and 50 ms. A following phase with contingency reversal eliminated the CS+/CS− differentiation at early and mid-latency intervals but reversed effects at the late interval. The authors interpret the effects at mid-latency and late intervals as enhanced processing of the pain-signalling CS+ in the auditory cortex. However, as electric shocks (UCS) were presented at 100, 175 and 250 ms after CS onset and source analysis was not performed, it remains unclear whether these Raf inhibitor effects reflect preferential auditory CS+ processing, a somatosensory CR (see above) or a mixture of both. Effects in the early interval were interpreted

as reflecting preferential auditory sensory processing of the safety signalling PLX3397 order CS− tones. A post hoc analysis of N1m interval identified by Kluge et al. (2011; 85–115 ms) revealed very similar results as our 100–150 ms interval analysis i.e. relatively enhanced CS− processing at the left temporoparietal junction and left occipitocerebral junction. An Masitinib (AB1010) analysis of their P2m (180–270 ms) interval in fact showed indications for enhanced CS+ processing but at bihemispheric somatosensory and right hemispheric parietal but not auditory sensory regions. Again, the N1m and P2m CS+ processing identified by Kluge and co-workers may at least partly reflect a conditioned response. The late effect might, however, additionally represent some form of conscious CS+ processing depending on contingency awareness which might have contributed to the unique reversal effects in this late component. Please note that, in the above classical conditioning studies, CS stimuli have been paired several hundred times with or without US and most subjects should have

been at least partly aware of the reinforcement plan, whereas absence of contingency awareness is one important factor within this MultiCS conditioning study. Importantly, the only interval which should not be superposed by a conditioned response (20–50 ms) revealed enhanced processing for safety signalling CS− identical to our study. The length of the CS stimuli (200 ms in Moses et al., 2010; and 250 ms in Kluge et al., 2011) is another important difference between these previous classical and our MultiCS conditioning studies (20 ms CS length). Although a differentiation of CS+ and CS− tones should be available immediately after CS onset, as our results would suggest, it remains unclear how differential processing of the subsequent parts of the auditory CS superimpose (e.

4 μM h−1), suggesting that N22-7 cells grown in the presence of MnO2 were deficient in the MnO2 reduction. When either oxygen, fumarate, nitrate, or dimethyl sulfoxide was used as an electron acceptor, N22-7 grew as fast as WT (data not shown). Besides, in MFCs, N22-7 generated a same level of current as WT, indicating that N22-7 retains the EET ability as that in WT (Fig. 2). These results suggest that Tn insertion in N22-7 click here resulted in a defect in a function specifically necessary for MnO2 reduction. Inverse-PCR and sequence analyses revealed that mini-Tn10 was inserted into SO3030, which is located in a gene cluster encoding a putative hydroxamate-type siderophore

biosynthesis system (SO3030 to SO3034). A deduced amino acid sequence of SO3030 showed the closest homology to a dihydroxamate siderophore (putrebactin) biosynthesis protein PubA (83% identity) in Shewanella sp. MR-4 (Kadi et al., 2008). Siderophores are high-affinity iron-chelating compounds secreted by organisms and are known to play important roles in iron acquisition (Wandersman & Delepelaire, 2004). In S. oneidensis MR-1, it has been reported that Selleckchem BMS-354825 siderophores

are not involved in Fe(III) solubilization during anaerobic Fe(III)-oxide respiration (Fennessey et al., 2010). However, roles of siderophores in anaerobic respiration of other metals, including MnO2, have not yet been investigated. To confirm that the disruption of SO3030 was responsible for the decreased MnO2-reduction rate of strain N22-7, an in-frame deletion mutant of SO3030 (ΔSO3030) and a plasmid-complemented strain ΔSO3030(pBBR-3030) were constructed, and their abilities to reduce MnO2 were compared with that of WT. We found that initial reduction rates of ΔSO3030 (123 ± 11 μM h−1) and ΔSO3030(pBBR-3030) (218 ± 3 μM h−1) were 53% and 95%, respectively, of that of WT, demonstrating 3-oxoacyl-(acyl-carrier-protein) reductase that the disruption of the SO3030 gene caused the decreased MnO2-reduction rate of strain N22-7. In contrast, an iron-oxide reduction by ΔSO3030 was as fast as that by WT (data not shown); a similar result has also been reported for an SO3031 knockout

mutant (Fennessey et al., 2010). To confirm that SO3030 is involved in the production of siderophore (putrebactin; Kadi et al., 2008), culture supernatants of WT and ΔSO3030 were subjected to the LC-MS analyses. It was found that a peak at m/z 373.1, which corresponds to the protonated ion of putrebactin (Kadi et al., 2008), was detected in WT (Fig. 3a), but not from the ΔSO3030 extract (Fig. 3b). It was also detected in a culture supernatant of ΔSO3030(pBBR-3030) (data not shown). These results suggest that MR-1 most likely produces putrebactin, a siderophore produced by Shewanella putrefaciens strain 200 (Ledyard & Butler, 1997) and Shewanella sp. MR-4 (Kadi et al., 2008), and SO3030 is essential for its biosynthesis. We next investigated whether or not the siderophore deficiency affected intracellular iron contents.

We developed an alternative method to sequencing, focusing on the SSR8 locus (constituted by GGT triplets from three to six repeats). The approach is based on asymmetric quantitative PCR, followed by high-resolution melting analysis with unlabelled probes (UP-HRM). PKC activation Data showed perfect concordance between

direct sequencing and UP-HRM, which is faster, simpler and more cost effective. Mycobacterium avium subsp. paratuberculosis (Map) is the causative agent of paratuberculosis in ruminants and other species. This bacterium is characterized by a very slow growth rate and limited genomic diversity (Stevenson et al., 2009). Numerous methods have been proposed to sub-type Map strains, such as multiplex PCR for IS900 integration loci, IS900 restriction fragment length polymorphism, amplified fragment length polymorphism, pulsed field gel electrophoresis and genotyping microarrays (Motiwala et al., 2006; Pribylova et al., 2009; Stevenson et al., 2009). However, methods based on micro- and minisatellite analyses are the most widely used techniques in this regard (Motiwala AG 14699 et al., 2006; Thibault et al., 2007) because of their relative simplicity and efficacy. Short sequence repeat (SSR) loci, particularly SSR1, SSR2, SSR8 and SSR9, showed highest allelic diversity, making these loci very useful for the evaluation of discriminatory indices (Amonsin et al., 2004; Thibault et al.,

2008). However, at present, the only method available for the scanning of these loci is direct sequencing, which requires expensive systems and dedicated

facilities. To overcome this problem, we developed a new alternative approach to sequencing, for the identification of SSR loci repeat number. We focused on the SSR8 locus, which comprises GGT triplets. So far, four alleles (ranging from three to six repeats) have been described for this locus (Amonsin et al., 2004; Ghadiali et al., 2004; Motiwala et al., 2006; Thibault et al., 2008). The new method is based on an asymmetric quantitative PCR (qPCR), followed by high-resolution melting (HRM) analysis with unlabelled probes (hereafter UP-HRM) (Zhou et al., 2004). The efficiency and specificity of the asymmetric PCR reaction was LY294002 improved by designing primers according to the linear-after-the-exponential PCR (LATE-PCR) strategy (Pierce et al., 2005), whereas to avoid any elongation during the amplification, the unlabelled probe was blocked at its 3′ end. The shortness of the probe (32 bp) enhanced the ability of HRM analysis to differentiate between sequences with very similar melting temperature (Tm), allowing clear identification of typical Tm values for every single allele. Map strains were collected at the Italian National Reference Centre for Paratuberculosis. Briefly, DNA was extracted by suspending one colony of Map in 100 μL of PCR-grade water.