Search

Leptospirosis is the leading zoonotic disease in terms of morbidity and mortality worldwide. Effective prevention is urgently needed as the drivers of disease transmission continue to intensify. The key challenge has been developing a widely applicable vaccine that protects against the >300 serovars that can cause leptospirosis. Live attenuated mutants are enticing vaccine candidates and poorly explored in the field. We evaluated a recently characterized motility-deficient mutant lacking the expression of a flagellar protein, FcpA. Although the fcpA- mutant has lost its ability to cause disease, transient bacteremia was observed. In two animal models, immunization with a single dose of the fcpA- mutant was sufficient to induce a robust anti-protein antibodies response that promoted protection against infection with different pathogenic Leptospira species. Furthermore, characterization of the immune response identified a small repertoire of biologically relevant proteins that are highly conserved among pathogenic Leptospira species and potential correlates of cross-protective immunity.

Bovine digital dermatitis (BDD) is a multifactorial polymicrobial infectious disease associated with multiple species and phylotypes of treponemes. However, despite the abundance of molecular signatures for treponemes that are identified in bovine lesions, relatively few isolates are cultured, and even fewer have been characterized at the level of protein expression. Here we report the successful isolation and characterization of novel strains of T. brennaborense and T. phagedenis from cases of BDD in Iowa dairy cows, and compare them to a well characterized strain of T. phagedenis, and the type strain of the more recently recognized T. pedis. Propagation of T. brennaborense was only possible at room temperature in Cooked Meat Medium, and not in oral treponeme enrichment medium at 37 °C as used for T. phagedenis and T. pedis. A prominent and rapid motility is observed by T. brennaborense under dark-field microscopy. The highly motile T. brennaborense strain 11-3 has an identical enzymatic profile to that of the only other isolate of T. brennaborense to be cultured from a lesion of BDD. Outer membrane protein profiles of each strain were compared by 2-D gel electrophoresis, and the five most abundant proteins in each strain were identified by mass spectrometry. All identified proteins are predicted to have signal peptides. Results identified outer membrane proteins specific to each strain including predicted membrane lipoproteins, ABC transporters and, as yet, uncharacterized proteins. Collectively, our results provide for the identification and characterization of outer membrane components of multiple phylotypes of treponemes associated with BDD which can facilitate development of vaccines and diagnostics in our efforts to eradicate the disease.

Stranded California sea lions (Zalophus californianus) along the California coast have been diagnosed with leptospirosis every year since at least the 1980s. Between September 2010 and November 2011, we followed 14 stranded California sea lions that survived to release and evaluated antibiotic efficacy in eliminating leptospiruria (urinary shedding of leptospires). Leptospiruria was assessed by real-time PCR of urine and urine culture, with persistence assessed using longitudinally collected samples. Serum chemistry was used to assess recovery of normal renal function. Microscopic agglutination testing (MAT) was performed to assess serum anti-Leptospira antibody titers, and the MAT reactivity patterns were consistent with L. interrogans serovar Pomona infection frequently observed in this population. Animals were initially treated for 6 to 16 d (median = 10.5; mean = 10.8) with antibiotics from the penicillin family, with some receiving additional antibiotics to treat other medical conditions. All urine cultures were negative; therefore, the presence of leptospiruria was assessed using PCR. Leptospiruria continued beyond the initial course of penicillin family antibiotics in 13 of the 14 sea lions, beyond the last antibiotic dose in 11 of the 14 sea lions, beyond recovery of renal function in 13 of the 14 sea lions, and persisted for at least 8 to 86 d (median = 45; mean = 46.8). Five animals were released with no negative urine PCR results detected; thus, their total shedding duration may have been longer. Cessation of leptospiruria was more likely in animals that received antibiotics for a greater duration, especially if coverage was uninterrupted. Real-time PCR results indicate that an antibiotic protocol commonly used to treat leptospirosis in rehabilitating California sea lions does not eliminate leptospiruria. It is possible that antibiotic protocols given for a longer duration and/or including other antibiotics may be effective in eliminating leptospiruria. These results may have important human and animal health implications, especially in rehabilitation facilities, as Leptospira transmission may occur through contact with animals with persistent leptospiruria.

Several real-time PCR assays are currently used for detection of pathogenic Leptospira spp.; however, few methods have been described for the successful evaluation of clinical urine samples. This study reports a rapid assay for the detection of pathogenic Leptospira spp. in California sea lions Zalophus californianus using real-time PCR with primers and a probe targeting the lipL32 gene. The PCR assay had high analytic sensitivity-the limit of detection was 3 genome copies per PCR volume using L. interrogans serovar Pomona DNA and 100% analytic specificity; it detected all pathogenic leptospiral serovars tested and none of the non-pathogenic Leptospira species (L. biflexa and L. meyeri serovar Semaranga), the intermediate species L. inadai, or the non-Leptospira pathogens tested. Our assay had an amplification efficiency of 1.00. Comparisons between the real-time PCR assay and culture isolation for detection of pathogenic Leptospira spp. in urine and kidney tissue samples from California sea lions showed that samples were more often positive by real-time PCR than by culture methods. Inclusion of an internal amplification control in the real-time PCR assay showed no inhibitory effects in PCR negative samples. These studies indicated that our real-time PCR assay has high analytic sensitivity and specificity for the rapid detection of pathogenic Leptospira species in urine and kidney tissue samples.

Discusses the selection of material for vocal performances by high school students. Describes adolescent voices as young with little vocal technique and only partially developed or unused register. Includes considerations of breath management, tessitura, energy, subject matter, and language. Provides a list of songs meeting appropriate requirements. (DK)

Brucellosis is a worldwide disease of humans and livestock that is caused by a number of very closely related classical Brucella species in the alpha-2 subdivision of the Proteobacteria. We report the complete genome sequence of Brucella abortus field isolate 9-941 and compare it to those of Brucella suis 1330 and Brucella melitensis 16 M. The genomes of these Brucella species are strikingly similar, with nearly identical genetic content and gene organization. However, a number of insertion-deletion events and several polymorphic regions encoding putative outer membrane proteins were identified among the genomes. Several fragments previously identified as unique to either B. suis or B. melitensis were present in the B. abortus genome. Even though several fragments were shared between only B. abortus and B. suis, B. abortus shared more fragments and had fewer nucleotide polymorphisms with B. melitensis than B. suis. The complete genomic sequence of B. abortus provides an important resource for further investigations into determinants of the pathogenicity and virulence phenotypes of these bacteria.

Leptospires belong to a genus of parasitic bacterial spirochetes that have adapted to a broad range of mammalian hosts. Mechanisms of leptospiral molecular evolution were explored by sequence analysis of four genes shared by 38 strains belonging to the core group of pathogenic Leptospira species: L. interrogans, L. kirschneri, L. noguchii, L. borgpetersenii, L. santarosai, and L. weilii. The 16S rRNA and lipL32 genes were highly conserved, and the lipL41 and ompL1 genes were significantly more variable. Synonymous substitutions are distributed throughout the ompL1 gene, whereas nonsynonymous substitutions are clustered in four variable regions encoding surface loops. While phylogenetic trees for the 16S, lipL32, and lipL41 genes were relatively stable, 8 of 38 (20%) ompL1 sequences had mosaic compositions consistent with horizontal transfer of DNA between related bacterial species. A novel Bayesian multiple change point model was used to identify the most likely sites of recombination and to determine the phylogenetic relatedness of the segments of the mosaic ompL1 genes. Segments of the mosaic ompL1 genes encoding two of the surface-exposed loops were likely acquired by horizontal transfer from a peregrine allele of unknown ancestry. Identification of the most likely sites of recombination with the Bayesian multiple change point model, an approach which has not previously been applied to prokaryotic gene sequence analysis, serves as a model for future studies of recombination in molecular evolution of genes.

The intestinal spirochaete Brachyspira pilosicoli causes colitis in a wide variety of host species. Little is known about the structure or protein constituents of the B. pilosicoli outer membrane (OM). To identify surface-exposed proteins in this species, membrane vesicles were isolated from B. pilosicoli strain 95-1000 cells by osmotic lysis in d[H.sub.2]O followed by isopycnic centrifugation in sucrose density gradients. The membrane vesicles were separated into a high-density fraction (HDMV; [rho] = 1.18 g [cm.sup.-3]) and a low-density fraction (LDMV; [rho] = 1.12 g [cm.sup.-3]). Both fractions were free of flagella and soluble protein contamination. LDMV contained predominantly OM markers (lipo-oligosaccharide and a 29 kDa B. pilosicoli OM protein) and was used as a source of antigens to produce mAbs. Five B. pilosicoli-specific mAbs reacting with proteins with molecular masses of 23, 24, 35, 61 and 79 kDa were characterized. The 23 kDa protein was only partially soluble in Triton X-114, whereas the 24 and 35 kDa proteins were enriched in the detergent phase, implying that they were integral membrane proteins or lipoproteins. All three proteins were localized to the B. pilosicoli OM by immunogold labelling using specific mAbs. The gene encoding the abundant, surface-exposed 23 kDa protein was identified by screening a B. pilosicoli 95-1000 genome library with the mAb and was expressed in Escherichia coli. Sequence analysis showed that it encoded a unique lipoprotein, designated BmpC. Recombinant BmpC partitioned predominantly in the OM fraction of E. coil strain SOLR. The mAb to BmpC was used to screen a collection of 13 genetically heterogeneous strains of B. pilosicoli isolated from five different host species. Interestingly, only strain 95-1000 was reactive with the mAb, indicating that either the surface-exposed epitope on BmpC is variable between strains or that the protein is restricted in its distribution within B. pilosicoli.

Abstract Background The genome of Mycobacterium avium subspecies paratuberculosis (MAP) is remarkably homogeneous among the genomes of bovine, human and wildlife isolates. However, previous work in our laboratories with the bovine K-10 strain has revealed substantial differences compared to sheep isolates. To systematically characterize all genomic differences that may be associated with the specific hosts, we sequenced the genomes of three U.S. sheep isolates and also obtained an optical map. Results Our analysis of one of the isolates, MAP S397, revealed a genome 4.8 Mb in size with 4,700 open reading frames (ORFs). Comparative analysis of the MAP S397 isolate showed it acquired approximately 10 large sequence regions that are shared with the human M. avium subsp. hominissuis strain 104 and lost 2 large regions that are present in the bovine strain. In addition, optical mapping defined the presence of 7 large inversions between the bovine and ovine genomes (~ 2.36 Mb). Whole-genome sequencing of 2 additional sheep strains of MAP (JTC1074 and JTC7565) further confirmed genomic homogeneity of the sheep isolates despite the presence of polymorphisms on the nucleotide level. Conclusions Comparative sequence analysis employed here provided a better understanding of the host association, evolution of members of the M. avium complex and could help in deciphering the phenotypic differences observed among sheep and cattle strains of MAP. A similar approach based on whole-genome sequencing combined with optical mapping could be employed to examine closely related pathogens. We propose an evolutionary scenario for M. avium complex strains based on these genome sequences.