First Isolation of Leptospira Borgpetersenii Serovar Hardjobovis in Guanacos (Lama Guanicoe) in South America

Background Leptospirosis is the most widespread zoonotic disease in the world. It is caused by pathogenic spirochetes of the genus Leptospira spp. and is maintained in nature through chronic renal infection of carrier animals, being rodents and other small mammals the main reservoirs. This bacterial genus is highly heterogeneous and divided into three clades (pathogenic, saprophyte and intermediate). Presence of pathogenic strains in wildlife populations is essential to monitor the epidemiological status of this disease worldwide. Methods In this study, we characterize an isolated strain of a Guanaco (Lama guanicoe) using Multiple Locus Variable number tandem repeats Analysis (MLVA) (Variable Number Tandem Repeats-VNTRs: 4, 7, 10, Lb4 and Lb5). To confirm the identity of the isolated strain, partial 16S rRNA sequencing was carried out. Phylogeny was constructed using Neighbor- joining. Results The pathogenic leptospiral strain isolated from Llama guanicoe had the genetic profile identical to L. borgpetersenii serovar Hardjobovis reference strain Sponselee. Conclusions To the best of our knowledge, this is the first isolation and genetic characterization of a pathogenic leptospiral strain in Guanacos in South America.

Of Mice, Cattle, and Men: A Review of the Eco-Epidemiology of Leptospira borgpetersenii Serovar Ballum

In New Zealand (NZ), leptospirosis is a mostly occupational zoonosis, with >66% of the recently notified cases being farm or abattoir workers. Livestock species independently maintain Leptospira borgpetersenii serovar Hardjo and L. interrogans serovar Pomona, and both are included in livestock vaccines. The increasing importance in human cases of Ballum, a serovar associated with wildlife, suggests that wildlife may be an overlooked source of infection. Livestock could also act as bridge hosts for humans. Drawing from disease ecology frameworks, we chose five barriers to include in this review based on the hypothesis that cattle act as bridge hosts for Ballum. Using a narrative methodology, we collated published studies pertaining to (a) the distribution and abundance of potential wild maintenance hosts of Ballum, (b) the infection dynamics (prevalence and pathogenesis) in those same hosts, (c) Ballum shedding and survival in the environment, (d) the exposure and competency of cattle as a potential bridge host, and (e) exposure for humans as a target host of Ballum. Mice (Mus musculus), rats (Rattus rattus, R. norvegicus) and hedgehogs (Erinaceus europaeus) were suspected as maintenance hosts of Ballum in NZ in studies conducted in the 1970s–1980s. These introduced species are distributed throughout NZ, and are present on pastures. The role of other wildlife in Ballum (and more broadly Leptospira) transmission remains poorly defined, and has not been thoroughly investigated in NZ. The experimental and natural Ballum infection of cattle suggest a low pathogenicity and the possibility of shedding. The seroprevalence in cattle appears higher in recent serosurveys (3 to 14%) compared with studies from the 1970s (0 to 3%). This review identifies gaps in the knowledge of Ballum, and highlights cattle as a potential spillover host. Further studies are required to ascertain the role that wild and domestic species may play in the eco-epidemiology of Ballum in order to understand its survival in the environment, and to inform control strategies.

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).

Aseptic meningitis as the initial presentation of Leptospira borgpetersenii serovar Tarassovi: two case reports and a literature review

Background Leptospirosis is a zoonotic illness caused by pathogenic spirochetes of the genus Leptospira. The disease spectrum ranges from a mild influenza-like presentation to a more serious Weil’s syndrome. Leptospirosis rarely presents as a primary neurological syndrome. We report two cases of Leptospira borgpetersenii serovar Tarasssovi presenting as aseptic meningitis in Sri Lanka. Case presentation We describe case reports of two patients presenting as symptomatic aseptic meningitis due to neuroleptospirosis. Both patients had significant neurological involvement at presentation in the absence of common clinical features of leptospirosis. These patients were initially managed as bacterial or viral meningitis and leptospirosis was suspected due to a history of exposure to contaminated water. Subsequently, they were diagnosed to have neuroleptospirosis by positive Leptospira serology and both patients gained full recovery. Conclusion Our report highlights the importance of considering leptospirosis as a differential diagnosis in patients with aseptic meningitis in endemic settings. Obtaining a detailed occupational and recreational history is helpful in diagnosing neuroleptospirosis promptly. We report the association of Leptospira borgpetersenii serovar (sv.) Tarassovi (strain bakeri) in causing aseptic meningitis, which has not been reported to the best of our knowledge.

Molecular and serological epidemiology of Leptospira infection in cats in Okinawa Island, Japan

Leptospirosis is a zoonosis caused by pathogenic Leptospira spp. Cats have been reported to be infected with Leptospira spp. and shed the bacteria in the urine. However, the importance of cats as an infection source for humans remains unclear. In this study, Leptospira infection in cats in Okinawa Prefecture, Japan, where leptospirosis is endemic, was investigated by leptospiral antibody and DNA detection using microscopic agglutination test and nested PCR, respectively. Moreover, multilocus sequence typing (MLST) and whole genome sequencing (WGS) were conducted on the Leptospira borgpetersenii serogroup Javanica isolated from cats, black rats, a mongoose, and humans. Anti-Leptospira antibodies were detected in 16.6% (40/241) of the cats tested, and the predominant reactive serogroup was Javanica. The leptospiral flaB gene was detected in 7.1% (3/42) of cat urine samples, and their sequences were identical and identified as L. borgpetersenii. MLST and WGS revealed the genetic relatedness of L. borgpetersenii serogroup Javanica isolates. This study indicated that most seropositive cats had antibodies against the serogroup Javanica and that cats excreted L. borgpetersenii in the urine after infection. Further, genetic relatedness between cat and human isolates suggests that cats may be a maintenance host for L. borgpetersenii serogroup Javanica and a source for human infection.

Distinct transcriptional profiles of Leptospira borgpetersenii serovar Hardjo strains JB197 and HB203 cultured at different temperatures

Background Leptospirosis is a zoonotic, bacterial disease, posing significant health risks to humans, livestock, and companion animals around the world. Symptoms range from asymptomatic to multi-organ failure in severe cases. Complex species-specific interactions exist between animal hosts and the infecting species, serovar, and strain of pathogen. Leptospira borgpetersenii serovar Hardjo strains HB203 and JB197 have a high level of genetic homology but cause different clinical presentation in the hamster model of infection; HB203 colonizes the kidney and presents with chronic shedding while JB197 causes severe organ failure and mortality. This study examines the transcriptome of L. borgpetersenii and characterizes differential gene expression profiles of strains HB203 and JB197 cultured at temperatures during routine laboratory conditions (29°C) and encountered during host infection (37°C). Methodology/Principal findings L. borgpetersenii serovar Hardjo strains JB197 and HB203 were isolated from the kidneys of experimentally infected hamsters and maintained at 29°C and 37°C. RNAseq revealed distinct gene expression profiles; 440 genes were differentially expressed (DE) between JB197 and HB203 at 29°C, and 179 genes were DE between strains at 37°C. Comparison of JB197 cultured at 29°C and 37°C identified 135 DE genes while 41 genes were DE in HB203 with those same culture conditions. The consistent DE of ligB, which encodes the outer membrane virulence factor LigB, was validated by immunoblotting and 2D-DIGE. Differential expression of lipopolysaccharide was also observed between JB197 and HB203. Conclusions/Significance Investigation of the L. borgpetersenii JB197 and HB203 transcriptome provides unique insight into the mechanistic differences between acute and chronic disease. Characterizing the nuances of strain to strain differences and investigating the environmental sensitivity of Leptospira to temperature is critical to the development and progress of leptospirosis prevention and treatment technologies, and is an important consideration when serovars are selected and propagated for use as bacterin vaccines as well as for the identification of novel therapeutic targets.

Bovine Immune Response to Vaccination and Infection with Leptospira borgpetersenii Serovar Hardjo

ABSTRACT This study examined the humoral and cellular response of cattle vaccinated with two commercial leptospiral vaccines, Leptavoid and Spirovac, and a novel bacterin vaccine using Seppic Montanide oil emulsion adjuvant. Vaccination was followed by experimental challenge. All vaccinated cattle were protected from colonization of the kidney and shedding of Leptospira in urine, as detected by culture and immunofluorescence assay. Agglutinating antibody titers were detected in vaccinated cattle at 4 weeks following vaccination, with small anamnestic response detected following experimental challenge. Only animals vaccinated with the oil emulsion-adjuvanted bacterin produced significant IgG2 titers following vaccination, and nonvaccinated animals produced serum IgA titers after experimental challenge. CD4+ and γδ T cells from vaccinated cattle proliferated when cultured with antigen ex vivo. Cellular responses included a marked proliferation of γδ T cells immediately following experimental challenge in vaccinated cattle and release of gamma interferon (IFN-γ), interleukin 17a (IL-17a), and IL-12p40 from stimulated cells. Proliferative and cytokine responses were found not just in peripheral mononuclear cells but also in lymphocytes isolated from renal lymph nodes at 10 weeks following experimental challenge. Overall, effects of leptospirosis vaccination and infection were subtle, resulting in only modest activation of CD4+ and γδ T cells. The use of Seppic Montanide oil emulsion adjuvants may shorten the initiation of response to vaccination, which could be useful during outbreaks or in areas where leptospirosis is endemic. IMPORTANCE Leptospirosis is an underdiagnosed, underreported zoonotic disease of which domestic livestock can be carriers. As a reservoir host for Leptospira borgpetersenii serovar Hardjo, cattle may present with reproductive issues, including abortion, birth of weak or infected calves, or failure to breed. Despite years of study and the availability of commercial vaccines, detailed analysis of the bovine immune response to vaccination and Leptospira challenge is lacking. This study evaluated immunologic responses to two efficacious commercial vaccines and a novel bacterin vaccine using an adjuvant chosen for enhanced cellular immune responses. Antigen-specific responsive CD4 and γδ T cells were detected following vaccination and were associated with release of inflammatory cytokines IFN-γ and IL-17a after stimulation. CD4 and γδ cells increased in the first week after infection and, combined with serum antibody, may play a role in clearance of bacteria from the blood and resident tissues. Additionally, these antigen-reactive T cells were found in the regional lymph nodes following infection, indicating that memory responses may not be circulating but are still present in regional lymph nodes. The information gained in this study expands knowledge of bovine immune response to leptospirosis vaccines and infection. The use of oil emulsion adjuvants may enhance early immune responses to leptospiral bacterins, which could be useful in outbreaks or situations where leptospirosis is endemic.

Persistence of Leptospira borgpetersenii Serovar Hardjo in Refrigerated Raw Milk: A Transmission Risk of Leptospirosis to Humans

Leptospira borgpetersenii serovar Hardjo (LH) is an important infectious agent of reproduction pathologies and lactation decline in cattle, with a possible zoonotic role. To figure out the potential zoonotic risk for human raw-milk consumption, the present study aims at assessing the persistence and viability of LH in refrigerated raw milk over a 10-day period, which is set as the maximum time range for raw-milk domestic consumption. A negative sample of fresh raw milk was contaminated with an LH strain (2 × 108 Leptospires/mL) and analyzed by a rrs (16S) gene targeting real-time PCR (rPCR) protocol for LH DNA at days 1, 2, 3, 6, 7, 9, and 10. Seven aliquots of the same sampling time were inoculated into a semisolid EMJH media for bacterial culture. All aliquots tested positive in both rPCR and culture, which demonstrates that raw milk does not alter the detectability and viability of LH, respectively. The analytical sensitivity (LoD, limit of detection) determined for the rPCR (103 Leptospires/mL) was repeatable during the study, whereas it gradually decreased when it came to the bacterial culture. This study demonstrates that bovine raw milk might be a potential vehicle of infection by LH, even when storage conditions are strictly respected.