Crosstalk between E-Cadherin/β-Catenin and NF-κB Signaling Pathways: The Regulation of Host-Pathogen Interaction during Leptospirosis

Approximately 1 million cases of leptospirosis, an emerging infectious zoonotic disease, are reported each year. Pathogenic Leptospira species express leucine-rich repeat (LRR) proteins that are rarely expressed in non-pathogenic Leptospira species. The LRR domain-containing protein family is vital for the virulence of pathogenic Leptospira species. In this study, the biological mechanisms of an essential LRR domain protein from pathogenic Leptospira were examined. The effects of Leptospira and recombinant LRR20 (rLRR20) on the expression levels of factors involved in signal transduction were examined using microarray, quantitative real-time polymerase chain reaction, and western blotting. The secreted biomarkers were measured using an enzyme-linked immunosorbent assay. rLRR20 colocalized with E-cadherin on the cell surface and activated the downstream transcription factor β-catenin, which subsequently promoted the expression of MMP7, a kidney injury biomarker. Additionally, MMP7 inhibitors were used to demonstrate that the secreted MMP7 degrades surface E-cadherin. This feedback inhibition mechanism downregulated surface E-cadherin expression and inhibited the colonization of Leptospira. The degradation of surface E-cadherin activated the NF-κB signal transduction pathway. Leptospirosis-associated acute kidney injury is associated with the secretion of NGAL, a downstream upregulated biomarker of the NF-κB signal transduction pathway. A working model was proposed to illustrate the crosstalk between E-cadherin/β-catenin and NF-κB signal transduction pathways during Leptospira infection. Thus, rLRR20 of Leptospira induces kidney injury in host cells and inhibits the adhesion and invasion of Leptospira through the upregulation of MMP7 and NGAL.

First Isolation and Molecular Typing of Pathogenic and Intermediate Leptospira Species from Urine of Symptomatic Dogs

Aim of this study was to evaluate, the presence and diversity of Leptospira spp. in blood and urine samples collected from 175 owned-dogs from Sardinia, Italy. After determination of leptospiral infection by microscopic agglutination test (MAT), urine from MAT-positive dogs were examined by real-time polymerase chain reaction (lipL32 rt-PCR) and then isolated by culture. In order to characterize obtained serovars, positive cultures were then subjected to 16S rRNA and secY sequencing, phylogenetic analysis and Multilocus Sequence Typing (MLST). Results showed that seven dogs (4%; 95% CI: 0–55) had Leptospira DNAs in their urine and five strains were isolated from urine cultures. The three different sequence types (ST17, ST198 and ST24) belonging to Leptospira interrogans genomospecies identified by MLST analyses in this study, confirmed that the leptospiral infection was widespread in Sardinian dogs. We also reported the first characterization of a new Leptospira spp. isolated from urine of one dog living in the study area. Whole genome sequencing and phylogenetic analysis, confirmed that this genospecies was closely related to Leptospira hovindhougenii, an intermediate Leptospira spp. with unknown pathogenicity previously isolated from a rat in Denmark. Further studies are required to clarify whether healthy dogs that shed leptospires in their urine could represent a zoonotic risk for humans in this region.

Divergent lineages of pathogenic Leptospira species are widespread and persisting in the environment in Puerto Rico, USA

Background: Leptospirosis, caused by Leptospira bacteria, is a common zoonosis worldwide more prevalent in the tropics. Reservoir species and risk factors have been identified but surveys for environmental sources of leptospirosis are rare. Furthermore, understanding of environmental Leptospira containing pathogenic genes and possibly capable of causing disease is incomplete and could result in some pathogenic strains evading detection, thereby convoluting diagnosis, prevention, and epidemiology. Methodology/Principal Findings: 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 pathogenic Leptospira DNA 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. 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. Conclusions/Significance: Diverse and novel pathogenic Leptospira are widespread in the environment in Puerto Rico. The disease potential of the novel lineages is unknown but several persisted for >1 year in soil, which could contaminate water. This work increases understanding of environmental Leptospira and should improve leptospirosis surveillance and diagnostics.

Molecular biology as a diagnostic tool for detection of Leptospira spp. in cows of a border region – case report

The reproductive efficiency of livestock is the basis for the success of livestock, dairy or beef, and having high reproductive performance depends on several factors within the production system and the presence of infectious diseases of the reproductive sphere in the herd is one of the factors that can compromise that efficiency. The aim of this study was to use molecular biology as a diagnostic tool for the detection of Leptospira spp. DNA in cows with reproductive disorders on a rural property in the municipality of Boca do Acre, Amazonas, Brazil. Vaginal mucus was collected from nine Nelore breeding cows with a history of abortion and birth of weak calves submitted to DNA extraction and nested-PCR technique for 16S gene amplification at the bacterial genus level. Of the nine samples analyzed, five (55.55%) amplified a product of 331bp. The municipality of Boca do Acre is bordered by Peru and Bolivia, and knowledge of the prevalence of the disease, serovars, and circulating Leptospira species is essential for the adoption of measures related to animal husbandry, as well as health education for ranchers and their workers to avoid a possible occupational infection since this disease is considered an important zoonosis. New molecular studies using primers that allow the identification of the Leptospira species and mainly pathogenic species should be conducted in this region in order to elucidate the possible species of this etiological agent and the possible reservoirs of the disease to begin the understanding of the epidemiology of this disease in cattle in this region of border.

Leptospira infection in rural areas of Uraba region, Colombia: a prospective study

The objective of this study was to analyze the eco-epidemiological aspects of Leptospira seroprevalence and seroincidence, and its associated factors in two municipalities of northwest Colombia. A prospective study was performed in rural areas of Uraba, Antioquia, Colombia. The study enrolled 597 people between November 2015 and January 2016, of which 274 people were followed up one year later. Serologic testing was performed by a microscopic agglutination. The outcomes were seroprevalent and seroincident cases, and the main exposure was an outdoor occupation. A binary and mixed effect multinomial logistic regression model was used to estimate factors associated with seroprevalent or seroincident cases of Leptospira infection. The overall Leptospira seroprevalence was 27.81% (95%CI:23.62;32.49) and the overall cumulative seroincidence for Leptospira was 14.60% (95%CI:10.33;20.23). Multivariable analysis showed that factors associated with L. interrogans serogroups seropositivity were outdoor occupation, male gender, older age, the presence of dirt soil in the household, and the presence of piglets and opossums. It also showed that factors associated with other Leptospira species serogroups were the presence of pit latrines and of turkeys. In addition, the multivariable model of seroincident cases of L. interrogans serogroups evidenced outdoor occupations, the presence of rats, and corn cultivation as risk factors. Likewise, the multivariable model for seroincident cases of other Leptospira species showed that the presence of hunting canines and cassava cultivation were risk factors. We found specific factors associated with the transmission of Leptospira serogroups contribute to the understanding of the epidemiology of Leptospira infection in rural areas of Uraba, Colombia.

High Diversity of Leptospira Species Infecting Bats Captured in the Urabá Region (Antioquia-Colombia)

Leptospirosis is a globally distributed zoonotic disease caused by pathogenic bacteria of the genus Leptospira. This zoonotic disease affects humans, domestic animals and wild animals. Colombia is considered an endemic country for leptospirosis; Antioquia is the second department in Colombia, with the highest number of reported leptospirosis cases. Currently, many studies report bats as reservoirs of Leptospira spp. but the prevalence in these mammals is unknown. The goal of this study was to better understand the role of bats as reservoir hosts of Leptospira species and to evaluate the genetic diversity of circulating Leptospira species in Antioquia-Colombia. We captured 206 bats in the municipalities of Chigorodó (43 bats), Carepa (43 bats), Apartadó (39 bats), Turbo (40 bats), and Necoclí (41 bats) in the Urabá region (Antioquia-Colombia). Twenty bats tested positive for Leptospira spp. infection (20/206—9.70%) and the species of infected bats were Carollia perspicillata, Dermanura rava, Glossophaga soricina, Molossus molossus, Artibeus planirostris, and Uroderma convexum. These species have different feeding strategies such as frugivorous, insectivores, and nectarivores. The infecting Leptospira species identified were Leptospira borgpetersenii (3/20–15%), Leptospira alexanderi (2/20–10%), Leptospira noguchii (6/20–30%), Leptospira interrogans (3/20–15%), and Leptospira kirschneri (6/20–30%). Our results showed the importance of bats in the epidemiology, ecology, and evolution of Leptospira in this host-pathogen association. This is the first step in deciphering the role played by bats in the epidemiology of human leptospirosis in the endemic region of Urabá (Antioquia-Colombia).

High Diversity of Leptospira Species Infecting Bats Captured in the Uraba Region (Antioquia-Colombia)

Leptospirosis is a globally distributed zoonotic disease caused by pathogenic bacteria of the genus Leptospira. This zoonotic disease affects humans, domestic, or wild animals. Colombia is considered an endemic country for leptospirosis; and Antioquia is the second department in Colombia with the highest number of reported leptospirosis cases. Currently, many studies report bats as reservoirs of Leptospira spp. but its prevalence in these mammals is unknown. In the present study we aimed to better understand the role of bats as reservoir hosts of Leptospira species and to evaluate the genetic diversity of circulating Leptospira species in Antioquia-Colombia. We captured 206 bats in the municipalities of Chigorodó (43 bats), Carepa (43 bats), Apartadó (39 bats), Turbo (40 bats), and Necoclí (41 bats) in the Urabá region (Antioquia-Colombia). Twenty bats were positive for Leptospira spp. infection (20/206 - 9,70%) and the species of infected bats were Carollia perspicillata, Dermatura rava, Glossophaga soricina, Molossus molossus, Artibeus planirostris, and Uroderma convexum. These species have different feeding strategies such as frugivorous, insectivores, and nectarivores. The infecting Leptospira species identified were Leptospira borgpetersenii (3/20 – 15%), Leptospira alexanderi (2/20 – 10%), Leptospira noguchii (6/20 – 30%), Leptospira interrogans (3/2 – 15%), and Leptospira kirschneri (6/20 – 30%). The results of this research show the importance of bats in the epidemiology, ecology and evolution of Leptospira in this host-pathogen association. This is the first step in deciphering the role played by bats in the epidemiology of human leptospirosis in the endemic region of Uraba (Antioquia-Colombia).

Extracellular Proteome Analysis Shows the Abundance of Histidine Kinase Sensor Protein, DNA Helicase, Putative Lipoprotein Containing Peptidase M75 Domain and Peptidase C39 Domain Protein in Leptospira interrogans Grown in EMJH Medium

Leptospirosis is a re-emerging form of zoonosis that is caused by the spirochete pathogen Leptospira. Extracellular proteins play critical roles in the pathogenicity and survival of this pathogen in the host and environment. Extraction and analysis of extracellular proteins is a difficult task due to the abundance of enrichments like serum and bovine serum albumin in the culture medium, as is distinguishing them from the cellular proteins that may reach the analyte during extraction. In this study, extracellular proteins were separated as secretory proteins from the culture supernatant and surface proteins were separated during the washing of the cell pellet. The proteins identified were sorted based on the proportion of the cellular fractions and the extracellular fractions. The results showed the identification of 56 extracellular proteins, out of which 19 were exclusively extracellular. For those proteins, the difference in quantity with respect to their presence within the cell was found to be up to 1770-fold. Further, bioinformatics analysis elucidated characteristics and functions of the identified proteins. Orthologs of extracellular proteins in various Leptospira species were found to be closely related among different pathogenic forms. In addition to the identification of extracellular proteins, this study put forward a method for the extraction and identification of extracellular proteins.

New Insights on Leptospira Infections in a Canine Population from North Sardinia, Italy: A Sero-Epidemiological Study

Leptospirosis is a widespread zoonosis recognized as a re-emerging infectious disease in a wide variety of animal species, including humans and dogs. No data exist regarding the presence of Leptospira species in the canine population of Sardinia Island. This study reports the first sero-survey for leptospirosis in kennel and owned dogs from six areas of the north of Sardinia. Sera from 1296 dogs were tested by microscopic agglutination test (MAT) specific for nine different serovars that are known to be well widespread in the Mediterranean environment. Moreover, kidney homogenates from rodents collected from the study area were also analyzed by LipL32 real-time PCR and multi-locus sequence type (MLST) on the basis of the analysis of seven concatenated loci. A total of 13% of the examined sera (95%CI: 11–15) tested positive for one or more serovars of Leptospira MAT detected; antibodies for serogroup Icterohaemorrhagiae (57%; 95%CI: 49–65) were the most common, followed by serovars Bratislava (22%; 95%CI: 16–28), Canicola (14%; 95%CI: 9–19), and Grippotyphosa (7%; 95%CI: 3–11). MLST analyses on isolates from rodents identified L. interrogans and L. borgpetersenii genomospecies. Different serovars belonging to pathogenic Leptospira serogroups are circulating in dogs from the island. Moreover, data obtained from rodents, indicated that rodents likely act as reservoir of spirochetes. Further sero-epidemiological studies are needed in order to obtain data from other collection sites in Sardinia and to increase the information on Leptospira species circulating in this area.