Leptospirosis is a devastating zoonotic disease affecting people and animals across the globe. Pathogenic leptospires are excreted in urine of reservoir hosts which directly or indirectly leads to ...continued disease transmission, via contact with mucous membranes or a breach of the skin barrier of another host. Human fatalities approach 60,000 deaths per annum; though most vertebrates are susceptible to leptospirosis, complex interactions between host species and serovars of
Leptospira
can yield disease phenotypes that vary from asymptomatic shedding in reservoir hosts, to multi-organ failure in incidental hosts. Clinical symptoms of acute leptospirosis reflect the diverse range of pathogenic species and serovars that cause infection, the level of exposure, and the relationship of the pathogen with the given host. However, in all cases, pathogenic
Leptospira
are excreted into the environment via urine from reservoir hosts which are uniformly recognized as asymptomatic carriers. Therefore, the reservoir host serves as the cornerstone of persistent disease transmission. Although bacterin vaccines can be used to abate renal carriage and excretion in domestic animal species, there is an urgent need to advance our understanding of immune-mediated host–pathogen interactions that facilitate persistent asymptomatic carriage. This review summarizes the current understanding of host–pathogen interactions in the reservoir host and prioritizes research to unravel mechanisms that allow for colonization but not destruction of the host. This information is required to understand, and ultimately control, the transmission of pathogenic
Leptospira
.
The causative agent of leptospirosis includes multiple serovars and species of pathogenic leptospires that are excreted via urine from reservoir hosts of infection. Primary isolation takes weeks to ...months, and is limited to semi-solid media at 28-30 °C. Here we present an alternative media formulation, HAN, compared to commercially available EMJH and the more specialized T80/40/LH media formulations, in semi-solid and liquid compositions, for the primary isolation of two diverse species and serovars of pathogenic leptospires directly from host kidney tissue. All three media types supported the isolation and propagation of L. interrogans serovar Copenhageni strain IC:20:001 in semi-solid media at 29 °C. However, only HAN and T80/40/LH supported the growth of L. borgpetersenii serovar Hardjo strain HB15B203 at 29 °C. In addition, HAN supported primary isolation at 37 °C. Both T80/40/LH and HAN supported primary isolation of strain IC:20:001 in liquid media at 29 °C but only HAN supported growth of strain HB15B203 in liquid media, at both 29 and 37 °C. HAN media supports the primary isolation of fastidious pathogenic leptospires directly from infected host tissue at either 29 or 37 °C: this formulation represents a more defined media for the continued optimization of growth factors required to support the primary isolation of the large and diverse range of species and serovars within the genus Leptospira circulating within domestic and wild animal populations.
Leptospirosis, an emerging zoonotic disease with worldwide distribution, is caused by spirochetes belonging to the genus Leptospira. More than 500,000 cases of severe leptospirosis are reported ...annually, with >10% of these being fatal. Leptospires can survive for weeks in suitably moist conditions before encountering a new host. Reservoir hosts, typically rodents, exhibit little to no signs of disease but shed large numbers of organisms in their urine. Transmission occurs when mucosal surfaces or abraded skin come into contact with infected urine or urine-contaminated water or soil. In humans, leptospires can cause a variety of clinical manifestations, ranging from asymptomatic or mild fever to severe icteric (Weil's) disease and pulmonary haemorrhage. Currently, little is known about how Leptospira persist within a reservoir host. Prior in vitro studies have suggested that leptospires alter their transcriptomic and proteomic profiles in response to environmental signals encountered during mammalian infection. However, no study has examined gene expression by leptospires within a mammalian host-adapted state. To obtain a more faithful representation of how leptospires respond to host-derived signals, we used RNA-Seq to compare the transcriptome of L. interrogans cultivated within dialysis membrane chambers (DMCs) implanted into the peritoneal cavities of rats with that of organisms grown in vitro. In addition to determining the relative expression levels of "core" housekeeping genes under both growth conditions, we identified 166 genes that are differentially-expressed by L. interrogans in vivo. Our analyses highlight physiological aspects of host adaptation by leptospires relating to heme uptake and utilization. We also identified 11 novel non-coding transcripts that are candidate small regulatory RNAs. The DMC model provides a facile system for studying the transcriptional and antigenic changes associated with mammalian host-adaptation, selection of targets for mutagenesis, and the identification of previously unrecognized virulence determinants.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Leptospirosis, caused by Leptospira bacteria, is a common zoonosis worldwide, especially in the tropics. Reservoir species and risk factors have been identified but surveys for environmental sources ...are rare. Furthermore, understanding of environmental Leptospira containing virulence associated genes and possibly capable of causing disease is incomplete, which may convolute leptospirosis diagnosis, prevention, and epidemiology.
We collected environmental samples from 22 sites in Puerto Rico during three sampling periods over 14-months (Dec 2018-Feb 2020); 10 water and 10 soil samples were collected at each site. Samples were screened for DNA from potentially pathogenic Leptospira using the lipL32 PCR assay and positive samples were sequenced to assess genetic diversity. One urban site in San Juan was sampled three times over 14 months to assess persistence in soil; live leptospires were obtained during the last sampling period. Isolates were whole genome sequenced and LipL32 expression was assessed in vitro. We detected pathogenic Leptospira DNA at 15/22 sites; both soil and water were positive at 5/15 sites. We recovered lipL32 sequences from 83/86 positive samples (15/15 positive sites) and secY sequences from 32/86 (10/15 sites); multiple genotypes were identified at 12 sites. These sequences revealed significant diversity across samples, including four novel lipL32 phylogenetic clades within the pathogenic P1 group. Most samples from the serially sampled site were lipL32 positive at each time point. We sequenced the genomes of six saprophytic and two pathogenic Leptospira isolates; the latter represent a novel pathogenic Leptospira species likely belonging to a new serogroup.
Diverse and novel pathogenic Leptospira are widespread in the environment in Puerto Rico. The disease potential of these lineages is unknown but several were consistently detected for >1 year in soil, which could contaminate water. This work increases understanding of environmental Leptospira diversity and should improve leptospirosis surveillance and diagnostics.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
Leptospirosis is a quintessential one health disease of humans and animals caused by pathogenic spirochetes of the genus
Leptospira
. Intra- and interspecies transmission is dependent on 1) ...reservoir host animals in which organisms replicate and are shed in urine over long periods of time, 2) the persistence of spirochetes in the environment, and 3) subsequent human-animal-environmental interactions. The combination of increased flooding events due to climate change, changes in human-animal-environmental interactions as a result of the pandemic that favor a rise in the incidence of leptospirosis, and under-recognition of leptospirosis because of nonspecific clinical signs and severe signs that resemble COVID-19 represents a “perfect storm” for resurgence of leptospirosis in people and domestic animals. Although often considered a disease that occurs in warm, humid climates with high annual rainfall, pathogenic
Leptospira
spp have recently been associated with disease in animals and humans that reside in semiarid regions like the southwestern US and have impacted humans that have a wide spectrum of socioeconomic backgrounds. Therefore, it is critical that physicians, veterinarians, and public health experts maintain a high index of suspicion for the disease regardless of geographic and socioeconomic circumstances and work together to understand outbreaks and implement appropriate control measures. Over the last decade, major strides have been made in our understanding of the disease because of improvements in diagnostic tests, molecular epidemiologic tools, educational efforts on preventive measures, and vaccines. These novel approaches are highlighted in the companion Currents in One Health by Sykes et al,
AJVR
, September 2022.
Leptospirosis is a worldwide zoonosis caused by pathogenic species of the genus
. The recent application of CRISPR interference (CRISPRi) to
facilitates targeted gene silencing and provides a new ...tool to investigate pathogenic mechanisms of leptospirosis. CRISPRi relies on the expression of a catalytically "dead" Cas9 (dCas9) and a single-guide RNA (sgRNA). Previously, our group generated a LipL32 and a double LigA/LigB (LigAB) mutant, which, in the current study, are characterized by whole-cell proteomics in comparison with control leptospires harboring plasmid expressing dCas9 alone. Comparison of control and LigAB mutant leptospires identified 46 significantly differentially expressed (DE) proteins, including 27 proteins that were less abundant and 19 proteins that were more abundant in the LigAB mutant compared with the control. Comparison of the control and LipL32 mutant leptospires identified 243 DE proteins, of which 84 proteins were more abundant and 159 were less abundant in the LipL32 mutant strain. Significantly increased amounts of known virulence impactors and surface membrane receptors, including LipL45, LipL31, LigB, and LipL41, were identified. The virulence of LipL32 and LigAB mutants were evaluated in the hamster model of leptospirosis; the LigAB mutant was unable to cause acute disease although mutant leptospires could still be recovered from target organs, albeit at a significantly lower bacterial burden (<850 and <16-fold in liver and kidney, respectively, in comparison with control), indicating attenuation of virulence and a shift to chronic bacterial persistence. Notably, the LipL32 mutant displayed augmented virulence as evidenced by early onset of clinical symptoms and increased numbers of circulating foamy macrophages. Validation of LipL32 and LigAB mutants recovered from liver and kidney in the presence or absence of antibiotic selection revealed high plasmid stability and, by extension, gene silencing
. Collectively, this work emphasizes the advantages and feasibility of using CRISPRi technology to evaluate and characterize virulence factors of leptospires and their respective host-pathogen interactions in animal models of leptospirosis. Importantly, it also provides insight into the requirements of LigA and LigB for acute disease and explores the impact of silencing expression of
, which resulted in substantial changes in amounts of outer membrane proteins.
Rattus norvegicus is a natural reservoir host for pathogenic species of Leptospira. Experimentally infected rats remain clinically normal, yet persistently excrete large numbers of leptospires from ...colonized renal tubules via urine, despite a specific host immune response. Whilst persistent renal colonization and shedding is facilitated in part by differential antigen expression by leptospires to evade host immune responses, there is limited understanding of kidney and urinary proteins expressed by the host that facilitates such biological equilibrium. Urine pellets were collected from experimentally infected rats shedding leptospires and compared to urine from non-infected controls spiked with in vitro cultivated leptospires for analysis by 2-D DIGE. Differentially expressed host proteins include membrane metallo endopeptidase, napsin A aspartic peptidase, vacuolar H+ATPase, kidney aminopeptidase and immunoglobulin G and A. Loa22, a virulence factor of Leptospira, as well as the GroEL, were increased in leptospires excreted in urine compared to in vitro cultivated leptospires. Urinary IgG from infected rats was specific for leptospires. Results confirm differential protein expression by both host and pathogen during chronic disease and include markers of kidney function and immunoglobulin which are potential biomarkers of infection.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
Leptospirosis is an archetypal One Health problem as described in the companion Currents in One Health article in the October 2022 issue of the
Journal of the American Veterinary Medical ...Association
by Sykes et al. A thorough understanding of leptospirosis requires a detailed analysis of the elaborate interplay among pathogenic leptospiral strains, host species, and the environment. Such an understanding is required to inform appropriate preventative measures including vaccine design, prophylaxis efforts, educational programs that help to reduce exposure to pathogenic spirochetes, as well as policy development. Because of the complex epidemiology of leptospirosis, a One Health approach as defined by the One Health Initiative Task Force is critical—an approach that calls for “the collaborative efforts of multiple disciplines working locally, nationally, and globally, to attain optimal health for people, animals and our environment.” Over the last three decades, progressive advances in cutting-edge molecular typing techniques, as well as our ability to rapidly generate and share large amounts of sequence data through establishment and growth of databases, have been central to accelerating a One Health understanding of the epidemiology of leptospirosis. Nevertheless, our dependence on serotype information because of the serovar-specific nature of current vaccines means that laborious serotyping efforts continue. With the advent of new approaches such as mRNA vaccines that are based on lipopolysaccharide immunogens, sequence- and/or proteomics-based typing methods may replace these methods.
Leptospirosis is a global zoonotic disease. The causative agent, pathogenic Leptospira species, survives in the renal tubules of chronically infected hosts, from where leptospires are shed via urine ...into the environment. Infection of new hosts can present as an array of acute and chronic disease processes reflecting variations in host-pathogen interactions. The present study was designed to reproduce the carrier phase of infection in Rattus norvegicus, thus facilitating shedding of leptospires in urine. Leptospires shed in urine were collected for proteomic analysis because these organisms reflect a naturally virulent form of Leptospira associated with infection of new hosts. Experimentally infected rats remained clinically asymptomatic but shed leptospires in urine for several months at concentrations of up to 10⁷ leptospires/ml of urine. Proteomic analysis of rat urine-isolated leptospires compared to in vitro-cultivated leptospires confirmed differential protein and antigen expression, as demonstrated by two-dimensional gel electrophoresis and immunoblotting. Furthermore, while serum from chronically infected rats reacted with many antigens of in vitro-cultivated Leptospira, few antigens of rat urine-isolated Leptospira were reactive. Results confirm that differential protein expression by Leptospira during chronic infection facilitates its persistence in the presence of a specific host antibody response.
Two spirochetes (designated strains LGVF01 and LGVF02
T
) were isolated from soil samples in San Juan, Puerto Rico in HAN media after selection using a combination of ELISA, agar plating, and colony ...screening by Fluorescent Antibody Testing (FAT) and PCR for
lipL32
and
secY
. Isolates were helix-shaped, aerobic, fast-growing, and highly motile. Genome sequence analysis indicated that both strains should be classified as members of a novel species within the pathogenic (P1) clade of the genus
Leptospira
. The average nucleotide identity between the two strains was 99.2 %, but below 93.2 % when compared to any previously described leptospiral species. Serotyping of strain LGVF02
T
indicates that it does not belong within any serogroup of
Leptospira
suggesting it also represents a new serovar. Collectively, strains LGVF01 and LGVF02
T
represent a new species of pathogenic leptospires for which the name
Leptospira sanjuanensis
sp. nov. is proposed. The type strain is LGVF02
T
(=NVSL-LGVF02
T
=KIT0302
T
).
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