A peptide-mediated capture PCR for the detection of Mycobacterium avium subsp. paratuberculosis in bulk milk samples was developed and characterized. Capture of the organism was performed using peptide aMptD, which had been shown to bind to the M. avium subsp. Paratuberculosis (Johne's disease) is a chronic and incurable granulomatous enteritis of ruminants caused by Mycobacterium avium subsp. paratuberculosis (29). The disease occurs worldwide with increasing frequency (32) and has a considerable economic impact on the livestock industry (21). Calves are mostly infected in early life, with high shedding and clinical disease commonly occurring at 2 to 5 years of age (37). Previous attempts to eradicate the disease from infected herds have frequently failed, despite a high degree of organizational and financial efforts (3,26). This is most likely due to the ubiquitous presence of the pathogen in the environments of infected herds (39) and its very great tenacity (7,20,52).An economically feasible alternative to eradication would be a control program aiming at the early identification and removal of high shedders, thereby reducing environmental contamination and infectious pressure on the herd. High shedders are more likely to secrete M. avium subsp. paratuberculosis in milk (50), and in addition, in herds with high shedders the pathogen is more likely to enter the milk by fecal contamination (8). Therefore, bulk milk might be a suitable diagnostic substrate for such an approach. Since the general infrastructure for testing of bulk milk from farms (i.e., untreated raw milk) is established (25), regular testing of this milk for the presence of M. avium subsp. paratuberculosis could allow early detection of herds with high shedders. The most convenient and amenable methods for such detection of M. avium subsp. paratuberculosis DNA in milk are enrichment via immunomagnetic separation (17,28,38) and peptide-mediated capture (47) followed by PCR, as these methods can be adapted to high-throughput testing using standard laboratory automation.In the study presented here, we followed this approach, using the phage display-derived peptide aMptD (46) for the capture of M. avium subsp. paratuberculosis in milk samples. The aMptD peptide was shown to bind to the surface-exposed MptD protein of M. avium subsp. paratuberculosis (ORF 3733C) (31), which is part of an M. avium subsp. paratuberculosis-specific pathogenicity island (46), and therefore this method, in contrast to the previously described peptide-mediated capture assay (47), is based on a defined receptor-ligand interaction. Furthermore, we thoroughly elucidated the strain and species cross-specificity of peptide aMptD for M. avium subsp. paratuberculosis by performing competitive capture assays, and we determined the kinetics and affinity of the receptor-ligand interaction by surface plasmon resonance (SPR) (27) in BIAcore biosensor experiments. Finally, in order to

Three facultatively anaerobic endospore-forming bacteria were isolated from the rhizosphere of sunflowers grown in fields of Rio Grande do Sul State, Brazil. The designated type strain P26E was previously identified as a sunflower growth promoting bacterium and is able to fix nitrogen and to excrete ammonia. According to analyses of 16S rRNA gene sequences, P26E presented similarity values above 98.8% in relation to Paenibacillus azotifigens NF2-4-5, Paenibacillus graminis RSA19, Paenibacillus jilunlii Be17, Paenibacillus salinicaeni LAM0A28, and Paenibacillus sonchi X19-5. Phylogenetic reconstructions based on 16S rRNA gene and core proteome data showed that the strains P26E, P3E and P32E form a distinct clade, which did not include any type strain of the currently described Paenibacillus species. Also, genomic comparisons using average nucleotide identity (ANI), Orthologous ANI and in silico DNA-DNA hybridization revealed similarity ranges below the recommended thresholds when the three isolates from sunflower were compared to their close relatives. The DNA G + C content of strain P26E was determined to be 49.4 mol%. The major cellular fatty acids are anteiso-C and iso-C, representing about 58 and 14% of the total fatty acids in P26E, respectively. Based on different taxonomic genomic metrics, phylogeny, and phenotypic data, we propose that strain P26E (= DSM 102269 = BR10509) represents a novel species within the genus Paenibacillus, for which the name Paenibacillus helianthi sp. nov. is proposed.

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Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne's disease, a chronic granulomatous enteritis in ruminants. Furthermore, infections of humans with MAP have been reported and a possible association with Crohn's disease and diabetes type I is currently discussed. MAP owns large sequence polymorphisms (LSPs) that were exclusively found in this mycobacteria species. The relevance of these LSPs in the pathobiology of MAP is still unclear. The mptD gene (MAP3733c) of MAP belongs to a small group of functionally uncharacterized genes, which are not present in any other sequenced mycobacteria species. mptD is part of a predicted operon (mptABCDEF), encoding a putative ATP binding cassette-transporter, located on the MAP-specific LSP14. In the present study, we generated an mptD knockout strain (MAPΔmptD) by specialized transduction. In order to investigate the potential role of mptD in the host, we performed infection experiments with macrophages. By this, we observed a significantly reduced cell number of MAPΔmptD early after infection, indicating that the mutant was hampered with respect to adaptation to the early macrophage environment. This important role of mptD was supported in mouse infection experiments where MAPΔmptD was significantly attenuated after peritoneal challenge. Metabolic profiling was performed to determine the cause for the reduced virulence and identified profound metabolic disorders especially in the lipid metabolism of MAPΔmptD. Overall our data revealed the mptD gene to be an important factor for the metabolic adaptation of MAP required for persistence in the host.

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