Genetic analysis of a Treponema phagedenis locus encoding antigenic lipoproteins with potential for antigenic variation.

Digital dermatitis (DD) is a painful and debilitating claw disease in cattle. Spirochetes of the genus Treponema are found in high numbers in the lesions and are likely to be involved in the pathogenesis. The occurrence of Treponema phagedenis in DD lesions, especially near the interface of healthy and diseased tissue, suggests that this species contributes to the development and/or progression of the lesions. In this study we characterized a genetic locus in T. phagedenis that contains coding regions for three antigenic proteins, PrrA, VpsA, and VpsB. Comparative analysis of homologous loci from fifteen strains suggests that prrA may be transposed into or out of this locus. Alterations in the copy number of TA repeats within the putative promoter region may regulate VpsA/B expression. The vpsA and prrA genes occur in allelic variants in different T. phagedenis isolates and may provide one explanation for the antigenic variation observed in T. phagedenis DD isolates.

Conservation of the Host-Interacting Proteins Tp0750 and Pallilysin among Treponemes and Restriction of Proteolytic Capacity to Treponema pallidum.

The spirochete Treponema pallidum subsp. pallidum is the causative agent of syphilis, a chronic, sexually transmitted infection characterized by multiple symptomatic and asymptomatic stages. Although several other species in the genus are able to cause or contribute to disease, T. pallidum differs in that it is able to rapidly disseminate via the bloodstream to tissue sites distant from the site of initial infection. It is also the only Treponema species able to cross both the blood-brain and placental barriers. Previously, the T. pallidum proteins, Tp0750 and Tp0751 (also called pallilysin), were shown to degrade host proteins central to blood coagulation and basement membrane integrity, suggesting a role for these proteins in T. pallidum dissemination and tissue invasion. In the present study, we characterized Tp0750 and Tp0751 sequence variation in a diversity of pathogenic and nonpathogenic treponemes. We also determined the proteolytic potential of the orthologs from the less invasive species Treponema denticola and Treponema phagedenis. These analyses showed high levels of sequence similarity among Tp0750 orthologs from pathogenic species. For pallilysin, lower levels of sequence conservation were observed between this protein and orthologs from other treponemes, except for the ortholog from the highly invasive rabbit venereal syphilis-causing Treponema paraluiscuniculi. In vitro host component binding and degradation assays demonstrated that pallilysin and Tp0750 orthologs from the less invasive treponemes tested were not capable of binding or degrading host proteins. The results show that pallilysin and Tp0750 host protein binding and degradative capability is positively correlated with treponemal invasiveness.

Complete Genome Sequence of Leptospira interrogans Serovar Bratislava, Strain PigK151.

Leptospira interrogans serovar Bratislava infection occurs in multiple domestic and wildlife species and is associated with poor reproductive performance in swine and horses. We present the complete genome assembly of strain PigK151 comprising two chromosomes, CI (4.457 Mbp) and CII (358 kbp).

Host response to leptospira infection.

Pathogenic Leptospira has the capacity to infect a broad range of mammalian hosts. Leptospirosis may appear as an acute, potentially fatal infection in accidental hosts, or progress into a chronic, largely asymptomatic infection in natural maintenance hosts. The course that Leptospira infection follows is dependent upon poorly understood factors, but is heavily influenced by both the host species and bacterial serovar involved in infection. Recognition of pathogen-associated molecular patterns (PAMPs) by a variety of host pattern recognition receptors (PRRs) activates the host immune system. The outcome of this response may result in bacterial clearance, limited bacterial colonization of a few target organs, principally the kidney, or induction of sepsis as the host succumbs to infection and dies. This chapter describes current knowledge of how the host recognizes Leptospira and responds to infection using innate and acquired immune responses. Aspects of immune-mediated pathology and pathogen strategies to evade the host immune response are also addressed.

Development of a real-time PCR for the detection of pathogenic Leptospira spp. in California sea lions.

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.

Isolation of Leptospira from a phocid: acute renal failure and mortality from Leptospirosis in rehabilitated northern elephant seals (Mirounga angustirostris), California, USA.

During rehabilitation, acute renal failure due to leptospirosis occurred in eight male northern elephant seals (Mirounga angustirostris) that stranded along the central California coast in 2011. Characteristic histologic lesions including renal tubular degeneration, necrosis, and mineralization, and mild lymphoplasmacytic interstitial nephritis were noted in the six animals examined. Immunohistochemistry, bacterial culture, and PCR were positive in 2/3, 2/3, and 3/4 seals, respectively, and 6/8 had high serum antibody titers to Leptospira interrogans serovar pomona. Pulsed-field gel electrophoresis confirmed one isolate as serovar pomona. Variable number tandem repeat (VNTR) analysis showed both elephant seal isolates were identical to each other but distinct from those isolated from California sea lions (Zalophus californianus). The time from stranding to onset of azotemia was 1 to 38 (median=24) days, suggesting some seals were infected at the rehabilitation facility. Based on temporal and spatial incidence of infection, transmission among elephant seals likely occurred during rehabilitation. Molecular (VNTR) analysis of the two isolates indicates there is a unique L. interrogans serovar pomona genotype in elephant seals, and sea lions were not the source of infection prior to or during rehabilitation. This study confirms the susceptibility of northern elephant seals to leptospirosis, indicates intraspecies transmission during rehabilitation, and reports the first isolation and preliminary characterization of leptospires from elephant seals.

Biochemical and molecular characterization of Treponema phagedenis-like spirochetes isolated from a bovine digital dermatitis lesion.

BACKGROUND: Bovine papillomatous digital dermatitis (DD) is the leading cause of lameness in dairy cattle and represents a serious welfare and economic burden. Found primarily in high production dairy cattle worldwide, DD is characterized by the development of an often painful red, raw ulcerative or papillomatous lesion frequently located near the interdigital cleft and above the bulbs of the heel. While the exact etiology is unknown, several spirochete species have been isolated from lesion material. Four isolates of Treponema phagedenis-like spirochetes were isolated from dairy cows in Iowa. Given the distinct differences in host, environmental niche, and disease association, a closer analysis of phenotypic characteristics, growth characteristics, and genomic sequences of T. phagedenis, a human genitalia commensal, and the Iowa DD isolates was undertaken. RESULTS: Phenotypically, these isolates range from 8.0 to 9.7 µm in length with 6-8 flagella on each end. These isolates, like T. phagedenis, are strictly anaerobic, require serum and volatile fatty acids for growth, and are capable of fermenting fructose, mannitol, pectin, mannose, ribose, maltose, and glucose. Major glucose fermentation products produced are formate, acetate, and butyrate. Further study was conducted with a single isolate, 4A, showing an optimal growth pH of 7.0 (range of 6-8.5) and an optimal growth temperature of 40 °C (range of 29 °C-43 °C). Comparison of partial genomic contigs of isolate 4A and contigs of T. phagedenis F0421 revealed > 95% amino acid sequence identity with amino acid sequence of 4A. In silico DNA-DNA whole genome hybridization and BLAT analysis indicated a DDH estimate of >80% between isolate 4A and T. phagedenis F0421, and estimates of 52.5% or less when compared to the fully sequenced genomes of other treponeme species. CONCLUSION: Using both physiological, biochemical and genomic analysis, there is a lack of evidence for difference between T. phagedenis and isolate 4A. The description of Treponema phagedenis should be expanded from human genital skin commensal to include being an inhabitant within DD lesions in cattle.

Asymptomatic and chronic carriage of Leptospira interrogans serovar Pomona in California sea lions (Zalophus californianus).

Since 1970, periodic outbreaks of leptospirosis, caused by pathogenic spirochetes in the genus Leptospira, have caused morbidity and mortality of California sea lions (Zalophus californianus) along the Pacific coast of North America. Yearly seasonal epizootics of varying magnitude occur between the months of July and December, with major epizootics occurring every 3-5 years. Genetic and serological data suggest that Leptospira interrogans serovar Pomona is the infecting serovar and is enzootic in the California sea lion population, although the mechanism of persistence is unknown. We report asymptomatic carriage of Leptospira in 39% (33/85) of wild, free-ranging sea lions sampled during the epizootic season, and asymptomatic seroconversion with chronic asymptomatic carriage in a rehabilitated sea lion. This is the first report of asymptomatic carriage in wild, free-ranging California sea lions and the first example of seroconversion and asymptomatic chronic carriage in a sea lion. Detection of asymptomatic chronic carriage of Leptospira in California sea lions, a species known to suffer significant disease and mortality from the same Leptospira strain, goes against widely-held notions regarding leptospirosis in accidental versus maintenance host species. Further, chronic carriage could provide a mechanism for persistent circulation of Leptospira in the California sea lion population, particularly if these animals shed infectious leptospires for months to years.

Leptospira interrogans catalase is required for resistance to H2O2 and for virulence.

Pathogenic Leptospira spp. are likely to encounter higher concentrations of reactive oxygen species induced by the host innate immune response. In this study, we characterized Leptospira interrogans catalase (KatE), the only annotated catalase found within pathogenic Leptospira species, by assessing its role in resistance to H(2)O(2)-induced oxidative stress and during infection in hamsters. Pathogenic L. interrogans bacteria had a 50-fold-higher survival rate under H(2)O(2)-induced oxidative stress than did saprophytic L. biflexa bacteria, and this was predominantly catalase dependent. We also characterized KatE, the only annotated catalase found within pathogenic Leptospira species. Catalase assays performed with recombinant KatE confirmed specific catalase activity, while protein fractionation experiments localized KatE to the bacterial periplasmic space. The insertional inactivation of katE in pathogenic Leptospira bacteria drastically diminished leptospiral viability in the presence of extracellular H(2)O(2) and reduced virulence in an acute-infection model. Combined, these results suggest that L. interrogans KatE confers in vivo resistance to reactive oxygen species induced by the host innate immune response.

Methylation and in vivo expression of the surface-exposed Leptospira interrogans outer-membrane protein OmpL32.

Recent studies have revealed that bacterial protein methylation is a widespread post-translational modification that is required for virulence in selected pathogenic bacteria. In particular, altered methylation of outer-membrane proteins has been shown to modulate the effectiveness of the host immune response. In this study, 2D gel electrophoresis combined with MALDI-TOF MS identified a Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 protein, corresponding to ORF LIC11848, which undergoes extensive and differential methylation of glutamic acid residues. Immunofluorescence microscopy implicated LIC11848 as a surface-exposed outer-membrane protein, prompting the designation OmpL32. Indirect immunofluorescence microscopy of golden Syrian hamster liver and kidney sections revealed expression of OmpL32 during colonization of these organs. Identification of methylated surface-exposed outer-membrane proteins, such as OmpL32, provides a foundation for delineating the role of this post-translational modification in leptospiral virulence.

A Leptospira borgpetersenii serovar Hardjo vaccine induces a Th1 response, activates NK cells, and reduces renal colonization.

Chronic infection of cattle with Leptospira borgpetersenii serovar Hardjo reduces animal production through reproductive failure and presents a persistent health threat to workers in the animal industry. Cattle are maintenance hosts for serovar Hardjo, and development of vaccines that establish long-term protective immunity has been problematic; induction of high titers of anti-serovar Hardjo antibody does not appear to be protective. Rather, development of an antigen-specific Th1 response appears to be critical for limiting renal colonization and urinary shedding of bacteria. In this study we compared two monovalent killed bacterial cell vaccines to assess long-term (12 months) protection against live serovar Hardjo challenge. Although neither vaccine prevented infection, renal colonization and urinary shedding of bacteria were reduced compared to those of control animals. Increased proliferation of CD4(+), CD8(+), and γδ T cells from vaccinated, but not control, animals was detected. In addition, NK cells from vaccinated animals and from all animals following infection, when exposed to antigen ex vivo, demonstrated a gamma interferon (IFN-γ) recall response. We propose that programming NK cells to respond quickly to L. borgpetersenii serovar Hardjo infection may be an important step toward developing protective immunity.

Transcriptional response of Leptospira interrogans to iron limitation and characterization of a PerR homolog.

Leptospirosis is a globally significant zoonosis caused by Leptospira spp. Iron is essential for growth of most bacterial species. Since iron availability is low in the host, pathogens have evolved complex iron acquisition mechanisms to survive and establish infection. In many bacteria, expression of iron uptake and storage proteins is regulated by Fur. L. interrogans encodes four predicted Fur homologs; we have constructed a mutation in one of these, la1857. We conducted microarray analysis to identify iron-responsive genes and to study the effects of la1857 mutation on gene expression. Under iron-limiting conditions, 43 genes were upregulated and 49 genes were downregulated in the wild type. Genes encoding proteins with predicted involvement in inorganic ion transport and metabolism (including TonB-dependent proteins and outer membrane transport proteins) were overrepresented in the upregulated list, while 54% of differentially expressed genes had no known function. There were 16 upregulated genes of unknown function which are absent from the saprophyte L. biflexa and which therefore may encode virulence-associated factors. Expression of iron-responsive genes was not significantly affected by mutagenesis of la1857, indicating that LA1857 is not a global regulator of iron homeostasis. Upregulation of heme biosynthetic genes and a putative catalase in the mutant suggested that LA1857 is more similar to PerR, a regulator of the oxidative stress response. Indeed, the la1857 mutant was more resistant to peroxide stress than the wild type. Our results provide insights into the role of iron in leptospiral metabolism and regulation of the oxidative stress response, including genes likely to be important for virulence.

Global proteome analysis of Leptospira interrogans.

Comparative global proteome analyses were performed on Leptospira interrogans serovar Copenhageni grown under conventional in vitro conditions and those mimicking in vivo conditions (iron limitation and serum presence). Proteomic analyses were conducted using iTRAQ and LC-ESI-tandem mass spectrometry complemented with two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. A total of 563 proteins were identified in this study. Altered expression of 65 proteins, including upregulation of the L. interrogans virulence factor Loa22 and 5 novel proteins with homology to virulence factors found in other pathogens, was observed between the comparative conditions. Immunoblot analyses confirmed upregulation of 5 of the known or putative virulence factors in L. interrogans exposed to the in vivo-like environmental conditions. Further, ELISA analyses using serum from patients with leptospirosis and immunofluorescence studies performed on liver sections derived from L. interrogans-infected hamsters verified expression of all but one of the identified proteins during infection. These studies, which represent the first documented comparative global proteome analysis of Leptospira, demonstrated proteome alterations under conditions that mimic in vivo infection and allowed for the identification of novel putative L. interrogans virulence factors.

Variable nucleotide tandem-repeat analysis revealing a unique group of Leptospira interrogans serovar Pomona isolates associated with California sea lions.

Leptospira interrogans serovar Pomona isolates were compared by variable nucleotide tandem-repeat typing. Most cattle isolates grouped together, while isolates from pigs and wildlife were distributed across several groups. Significantly, California sea lion isolates formed a unique group, providing evidence that these animals are maintenance hosts of serovar Pomona.

Geographical dissemination of Leptospira interrogans serovar Pomona during seasonal migration of California sea lions.

Leptospirosis is one of the most widespread bacterial zoonoses in the world and affects most mammalian species. Although leptospirosis is well documented and characterized in terrestrial species, less information is available regarding the distribution and impact of leptospirosis in marine mammals. Additionally, the role of animal migrations on the geographical spread of leptospirosis has not been reported. Periodic epizootic outbreaks of acute leptospirosis among California sea lions (Zalophus californianus) have been reported since 1971. In this study, we collected samples from California sea lions stranded along the Pacific coast of North America during the most recent epidemic in 2004, and maintained leptospirosis surveillance of the California sea lion population along the California coast through 2007. Several isolates of Leptospira interrogans serovar Pomona were obtained from kidney and urine samples collected during this study, a finding consistent with serological evidence that California sea lions are persistently exposed to this leptospiral serovar. Combined, these data support a model whereby California sea lions are maintenance hosts for L. interrogans serovar Pomona, yet periodically undergo outbreaks of acute infection. During the 2004 outbreak, the incidence of new leptospirosis cases among California sea lions coincided with the seasonal movement of male sea lions from rookeries along the coast of central and southern California north as far as British Columbia. These data show that seasonal animal movement contributes to the distribution of leptospirosis across a large geographical region.

Identification of a divided genome for VSH-1, the prophage-like gene transfer agent of Brachyspira hyodysenteriae.

The Brachyspira hyodysenteriae B204 genome sequence revealed three VSH-1 tail genes, hvp31, hvp60, and hvp37, in a 3.6-kb cluster. The location and transcription direction of these genes relative to those of the previously described VSH-1 16.3-kb gene operon indicate that the gene transfer agent VSH-1 has a noncontiguous, divided genome.

Genetic diversity of the Leptospiral immunoglobulin-like (Lig) genes in pathogenic Leptospira spp.

Recent serologic, immunoprotection, and pathogenesis studies identified the Lig proteins as key virulence determinants in interactions of leptospiral pathogens with the mammalian host. We examined the sequence variation and recombination patterns of ligA, ligB, and ligC among 10 pathogenic strains from five Leptospira species. All strains were found to have intact ligB genes and genetic drift accounting for most of the ligB genetic diversity observed. The ligA gene was found exclusively in L. interrogans and L. kirschneri strains, and was created from ligB by a two-step partial gene duplication process. The aminoterminal domain of LigB and the LigA paralog were essentially identical (98.5+/-0.8% mean identity) in strains with both genes. Like ligB, ligC gene variation also followed phylogenetic patterns, suggesting an early gene duplication event. However, ligC is a pseudogene in several strains, suggesting that LigC is not essential for virulence. Two ligB genes and one ligC gene had mosaic compositions and evidence for recombination events between related Leptospira species was also found for some ligA genes. In conclusion, the results presented here indicate that Lig diversity has important ramifications for the selection of Lig polypeptides for use in diagnosis and as vaccine candidates. This sequence information will aid the identification of highly conserved regions within the Lig proteins and improve upon the performance characteristics of the Lig proteins in diagnostic assays and in subunit vaccine formulations with the potential to confer heterologous protection.

Conservation of the S10-spc-alpha locus within otherwise highly plastic genomes provides phylogenetic insight into the genus Leptospira.

S10-spc-alpha is a 17.5 kb cluster of 32 genes encoding ribosomal proteins. This locus has an unusual composition and organization in Leptospira interrogans. We demonstrate the highly conserved nature of this region among diverse Leptospira and show its utility as a phylogenetically informative region. Comparative analyses were performed by PCR using primer sets covering the whole locus. Correctly sized fragments were obtained by PCR from all L. interrogans strains tested for each primer set indicating that this locus is well conserved in this species. Few differences were detected in amplification profiles between different pathogenic species, indicating that the S10-spc-alpha locus is conserved among pathogenic Leptospira. In contrast, PCR analysis of this locus using DNA from saprophytic Leptospira species and species with an intermediate pathogenic capacity generated varied results. Sequence alignment of the S10-spc-alpha locus from two pathogenic species, L. interrogans and L. borgpetersenii, with the corresponding locus from the saprophyte L. biflexa serovar Patoc showed that genetic organization of this locus is well conserved within Leptospira. Multilocus sequence typing (MLST) of four conserved regions resulted in the construction of well-defined phylogenetic trees that help resolve questions about the interrelationships of pathogenic Leptospira. Based on the results of secY sequence analysis, we found that reliable species identification of pathogenic Leptospira is possible by comparative analysis of a 245 bp region commonly used as a target for diagnostic PCR for leptospirosis. Comparative analysis of Leptospira strains revealed that strain H6 previously classified as L. inadai actually belongs to the pathogenic species L. interrogans and that L. meyeri strain ICF phylogenetically co-localized with the pathogenic clusters. These findings demonstrate that the S10-spc-alpha locus is highly conserved throughout the genus and may be more useful in comparing evolution of the genus than loci studied previously.

Detection of pathogenic Leptospira bacteria in pinniped populations via PCR and identification of a source of transmission for zoonotic leptospirosis in the marine environment.

Leptospirosis, caused by the spirochete Leptospira, is a geographically widespread disease that affects a broad range of mammals, including marine mammals. Among pinniped populations, periodic epizootics of leptospirosis are responsible for significant die-offs. Along the west coast of North America, the most recent leptospirosis epizootic occurred in 2004, during which samples were collected from cases ranging from California to British Columbia. The primary objective of this study was to use this well-defined sample set to determine the feasibility of using PCR techniques to diagnose Leptospira infection among pinniped populations in comparison with diagnostic methodologies commonly used for marine mammals. Successful amplification was achieved from a variety of samples, including freshly collected urine, urine stored at -80 degrees C for less than 6 months, and kidney (freshly collected, frozen, and decomposed), as well as feces- and urine-contaminated sand collected in the vicinity of a live-stranded animal. Pathological examination of tissue collected from Leptospira-infected animals revealed the presence of leptospiral antigen in the kidneys. The use of species-specific primer pairs revealed a pattern of host specificity for Leptospira interrogans in sea lions and Leptospira kirschneri in elephant seals. These studies indicate PCR is a sensitive and specific diagnostic tool for the detection of Leptospira infection in pinnipeds and reveal a potential source for epizootic, enzootic, and zoonotic spread of leptospirosis in a marine environment.

Collateral effects of antibiotics: carbadox and metronidazole induce VSH-1 and facilitate gene transfer among Brachyspira hyodysenteriae strains.

Brachyspira hyodysenteriae is an anaerobic spirochete and the etiologic agent of swine dysentery. The genome of this spirochete contains a mitomycin C-inducible, prophage-like gene transfer agent designated VSH-1. VSH-1 particles package random 7.5-kb fragments of the B. hyodysenteriae genome and transfer genes between B. hyodysenteriae cells. The chemicals and conditions inducing VSH-1 production are largely unknown. Antibiotics used in swine management and stressors inducing traditional prophages might induce VSH-1 and thereby stimulate lateral gene transfer between B. hyodysenteriae cells. In these studies, VSH-1 induction was initially detected by a quantitative real-time reverse transcriptase PCR assay evaluating increased transcription of hvp38 (VSH-1 head protein gene). VSH-1 induction was confirmed by detecting VSH-1-associated 7.5-kb DNA and VSH-1 particles in B. hyodysenteriae cultures. Nine antibiotics (chlortetracycline, lincomycin, tylosin, tiamulin, virginiamycin, ampicillin, ceftriaxone, vancomycin, and florfenicol) at concentrations affecting B. hyodysenteriae growth did not induce VSH-1 production. By contrast, VSH-1 was detected in B. hyodysenteriae cultures treated with mitomycin C (10 microg/ml), carbadox (0.5 microg/ml), metronidazole (0.5 microg/ml), and H(2)O(2) (300 microM). Carbadox- and metronidazole-induced VSH-1 particles transmitted tylosin and chloramphenicol resistance determinants between B. hyodysenteriae strains. The results of these studies suggest that certain antibiotics may induce the production of prophage or prophage-like elements by intestinal bacteria and thereby impact intestinal microbial ecology.