Concurrent colonization of rodent kidneys with multiple species and serogroups of pathogenic Leptospira.

Rodents are important reservoir hosts of pathogenic leptospires in the US Virgin Islands. Our previous work determined that trapped rodents were colonized with Leptospira borgpetersenii serogroup Ballum (n = 48) and/or Leptospira kirschneri serogroup Icterohaemorrhagiae (n = 3). In addition, nine rodents appeared to be colonized with a mixed population comprising more than one species/serogroup. The aim of this study was to validate this finding by characterizing clonal isolates derived from cultures of mixed species. Cultures of presumptive mixed species (designated LR1, LR5, LR37, LR57, LR60, LR61, LR68, LR70, and LR72) were propagated in different media including Hornsby-Alt-Nally (HAN) media, incubated at both 29℃ and 37℃, and T80/40/LH incubated at 29℃. Polyclonal reference antisera specific for serogroup Ballum and Icterohaemorrhagiae were used to enrich for different serogroups followed by subculture on agar plates. Individual colonies were then selected for genotyping and serotyping. Of the nine cultures of mixed species/serogroups, a single clonal isolate was separated in five of them: L. borgpetersenii serogroup Ballum in LR1, LR5, and LR37, and L. kirschneri serogroup Icterohaemorrhagiae in LR60 and LR72. In four of the cultures with mixed species (LR57, LR61, LR68, and LR70), clonal isolates of both L. borgpetersenii serogroup Ballum and L. kirschneri serogroup Icterohaemorrhagiae were recovered. Our results definitively establish that rodents can be colonized with more than one species/serogroup of Leptospira concurrently. The identification and characterization of multiple species/serogroups of Leptospira from individual reservoir hosts of infection are essential to understand the epidemiology and transmission of disease to both human and domestic animal populations.IMPORTANCEPathogenic Leptospira, the causative agent of human and animal leptospirosis, comprise a diverse genus of species/serogroups which are inherently difficult to isolate from mammalian hosts due to fastidious growth requirements. Molecular evidence has indicated that reservoir hosts of Leptospira may shed multiple species concurrently. However, evidence of this phenomena by culture has been lacking. Culture is definitive and is essential for comprehensive characterization of recovered isolates by high-resolution genome sequencing and serotyping. In this work, a protocol using recently developed novel media formulations, in conjunction with reference antisera, was developed and validated to demonstrate the recovery of multiple species/serogroups of pathogenic Leptospira from the same host. The identification and characterization of multiple species/serogroups of Leptospira from individual reservoir hosts of infection are essential to understand the epidemiology and transmission of disease to both human and domestic animal populations.

DNA Capture and Enrichment: A Culture-Independent Approach for Characterizing the Genomic Diversity of Pathogenic Leptospira Species.

Because they are difficult to culture, obtaining genomic information from Leptospira spp. is challenging, hindering the overall understanding of leptospirosis. We designed and validated a culture-independent DNA capture and enrichment system for obtaining Leptospira genomic information from complex human and animal samples. It can be utilized with a variety of complex sample types and diverse species as it was designed using the pan-genome of all known pathogenic Leptospira spp. This system significantly increases the proportion of Leptospira DNA contained within DNA extracts obtained from complex samples, oftentimes reaching >95% even when some estimated starting proportions were <1%. Sequencing enriched extracts results in genomic coverage similar to sequenced isolates, thereby enabling enriched complex extracts to be analyzed together with whole genome sequences from isolates, which facilitates robust species identification and high-resolution genotyping. The system is flexible and can be readily updated when new genomic information becomes available. Implementation of this DNA capture and enrichment system will improve efforts to obtain genomic data from unculturable Leptospira-positive human and animal samples. This, in turn, will lead to a better understanding of the overall genomic diversity and gene content of Leptospira spp. that cause leptospirosis, aiding epidemiology and the development of improved diagnostics and vaccines.

Complete Genome Sequences of Two Pasteurella multocida Isolates from Seabirds.

Pasteurella multocida is one of the major causes of mass mortalities in wild birds. Here, we report the complete genome sequences of two P. multocida isolates from wild populations of two endangered seabird species, the Indian yellow-nosed albatrosses (Thalassarche carteri) and the northern rockhopper penguins (Eudyptes moseleyi).

Animals Exposed to Leptospira Serogroups Not Included in Bacterins in the United States and Puerto Rico.

Leptospirosis is a worldwide zoonotic disease. Pathogenic leptospires colonize the renal tubules and genital tract of animals and are excreted via urine. Transmission occurs via direct contact or through contaminated water or soil. The microscopic agglutination test (MAT) is the gold standard for the serodiagnosis of leptospirosis. The present study aims to evaluate animal exposure to Leptospira in the U.S. and Puerto Rico during the period 2018-2020. The presence of antibodies against pathogenic Leptospira spp. was assessed with the MAT according to the standards of the World Organisation for Animal Health. A total of 568 sera were submitted for diagnostic, surveillance, or import/export testing from the U.S. and Puerto Rico. Seropositivity (≥1:100) was 51.8% (294/568) with agglutinating antibodies found in 115 (39.1%) cattle, 84 (28.6%) exotic animals, 38 (12.9%) horses, 22 (7.5%) goats, 15 (5.1%) dogs, 11 (3.7%) swine, and 9 (3.1%) sheep. The most detected serogroups were Australis, Grippotyphosa, and Ballum. The results showed that animals were exposed to serogroups/serovars not included in commercial bacterins such as Ballum, Bratislava (only in swine vaccine), and Tarassovi. Our findings suggest that more studies should include culture and concomitant genotyping to reduce animal disease and zoonotic risk through efficacious vaccine and diagnostic strategies.

Leptospira borgpetersenii serovar Hardjo and Leptospira santarosai serogroup Pyrogenes isolated from bovine dairy herds in Puerto Rico.

Leptospirosis is one of the most common zoonotic diseases in the world and endemic in the Caribbean Islands. Bovine leptospirosis is an important reproductive disease. Globally, cattle are recognized as a reservoir host for L. borgpetersenii serovar Hardjo, which is transmitted via urine, semen, and uterine discharges, and can result in abortion and poor reproductive performance. The dairy industry in Puerto Rico comprises up to 25% of agriculture-related income and is historically the most financially important agricultural commodity on the island. In this study, we report the isolation of two different pathogenic Leptospira species, from two different serogroups, from urine samples collected from dairy cows in Puerto Rico: L. borgpetersenii serogroup Sejroe serovar Hardjo and L. santarosai serogroup Pyrogenes. Recovered isolates were classified using whole-genome sequencing, serotyping with reference antisera and monoclonal antibodies, and immunoblotting. These results demonstrate that dairy herds in Puerto Rico can be concurrently infected with more than one species and serovar of Leptospira, and that bacterin vaccines and serologic diagnostics should account for this when applying intervention and diagnostic strategies.

Bovine Leptospirosis Due to Persistent Renal Carriage of Leptospira borgpetersenii Serovar Tarassovi.

Leptospirosis is a global zoonotic disease that causes significant morbidity and mortality in human and animal populations. Leptospira interrogans is a leading cause of human disease, and L. borgpetersenii is a leading cause of animal disease. Cattle are reservoir hosts of L. borgpetersenii serovar Hardjo, which is transmitted via urine, semen, and uterine discharges resulting in abortion and poor reproductive performance. Bovine bacterin vaccines can only protect against those serovars included in vaccine formulations and typically include serovar Hardjo among others. Genotyping and serotyping represent two different and unique methods for classifying leptospires that do not always correlate well; comprehensive characterization using either method requires recovery of isolates from infected animals. In this study, we report for the first time, isolation of L. borgpetersenii serovar Tarassovi from the urine of a dairy cow in the U.S. The classification of the isolate, designated strain MN900, was confirmed by whole-genome sequencing, serotyping with reference antisera and monoclonal antibodies, Matrix Assisted Laser Desorption/Ionization (MALDI), and immunoblotting with reference antisera. Strain MN900 was excreted in urine samples for 18 weeks even as the cow was seronegative for serovar Tarassovi. Strain MN900 has an unusual morphology since it is not as motile as other leptospires and lacks hooked ends. Serovar Tarassovi is not included in U.S. bacterin vaccines. These results demonstrate the importance of culture and concomitant genotyping and serotyping to accurately classify leptospires, and as required to design efficacious vaccine and diagnostic strategies to not only limit animal disease but reduce zoonotic risk.

Assessing rodents as carriers of pathogenic Leptospira species in the U.S. Virgin Islands and their risk to animal and public health.

Leptospirosis is a global zoonotic disease caused by pathogenic bacteria of the genus Leptospira. We sought to determine if rodents in U.S. Virgin Islands (USVI) are carriers of Leptospira. In total, 140 rodents were sampled, including 112 Mus musculus and 28 Rattus rattus. A positive carrier status was identified for 64/140 (45.7%); 49 (35.0%) were positive by dark-field microscopy, 60 (42.9%) by culture, 63 (45.0%) by fluorescent antibody testing, and 61 (43.6%) by real-time polymerase chain reaction (rtPCR). Molecular typing indicated that 48 isolates were L. borgpetersenii and 3 were L. kirschneri; the remaining nine comprised mixed species. In the single culture-negative sample that was rtPCR positive, genotyping directly from the kidney identified L. interrogans. Serotyping of L. borgpetersenii isolates identified serogroup Ballum and L. kirschneri isolates as serogroup Icterohaemorrhagiae. These results demonstrate that rodents are significant Leptospira carriers and adds to understanding the ecoepidemiology of leptospirosis in USVI.

Isolation of Leptospira kirschneri serovar Grippotyphosa from a red panda (Ailurus fulgens) after antimicrobial therapy: Case report.

A 1-year-old female red panda started showing symptoms of illness, including lethargy, anorexia, abdominal discomfort, and vomiting, shortly after transfer to a new zoo. Serum was tested for leptospirosis using the microscopic agglutination test, and a titer of 1:25,600 to serogroup Grippotyphosa was detected. Antimicrobial treatment with doxycycline was initiated. After completion of treatment and resolution of clinical symptoms, a urine sample was collected to ensure clearance of leptospires and cessation of urinary shedding prior to co-housing with other red pandas. A repeat serum sample taken 13 days later had a lower titer of 1:6,400 to serogroup Grippotyphosa. A sample of the animal's urine was cultured in HAN media and was culture positive for Leptospira. The recovered isolate was completely characterized by whole genome sequencing and serotyping with reference antisera, and the isolate was classified as Leptospira kirschneri serogroup Grippotyphosa serovar Grippotyphosa strain RedPanda1.

Mongooses (Urva auropunctata) as reservoir hosts of Leptospira species in the United States Virgin Islands, 2019-2020.

During 2019-2020, the Virgin Islands Department of Health investigated potential animal reservoirs of Leptospira spp., the bacteria that cause leptospirosis. In this cross-sectional study, we investigated Leptospira spp. exposure and carriage in the small Indian mongoose (Urva auropunctata, syn: Herpestes auropunctatus), an invasive animal species. This study was conducted across the three main islands of the U.S. Virgin Islands (USVI), which are St. Croix, St. Thomas, and St. John. We used the microscopic agglutination test (MAT), fluorescent antibody test (FAT), real-time polymerase chain reaction (lipl32 rt-PCR), and bacterial culture to evaluate serum and kidney specimens and compared the sensitivity, specificity, positive predictive value, and negative predictive value of these laboratory methods. Mongooses (n = 274) were live-trapped at 31 field sites in ten regions across USVI and humanely euthanized for Leptospira spp. testing. Bacterial isolates were sequenced and evaluated for species and phylogenetic analysis using the ppk gene. Anti-Leptospira spp. antibodies were detected in 34% (87/256) of mongooses. Reactions were observed with the following serogroups: Sejroe, Icterohaemorrhagiae, Pyrogenes, Mini, Cynopteri, Australis, Hebdomadis, Autumnalis, Mankarso, Pomona, and Ballum. Of the kidney specimens examined, 5.8% (16/270) were FAT-positive, 10% (27/274) were culture-positive, and 12.4% (34/274) were positive by rt-PCR. Of the Leptospira spp. isolated from mongooses, 25 were L. borgpetersenii, one was L. interrogans, and one was L. kirschneri. Positive predictive values of FAT and rt-PCR testing for predicting successful isolation of Leptospira by culture were 88% and 65%, respectively. The isolation and identification of Leptospira spp. in mongooses highlights the potential role of mongooses as a wildlife reservoir of leptospirosis; mongooses could be a source of Leptospira spp. infections for other wildlife, domestic animals, and humans.

Exposure and Carriage of Pathogenic Leptospira in Livestock in St. Croix, U.S. Virgin Islands.

From 2019-2020, the Virgin Islands Department of Health (VIDOH) investigated potential animal reservoirs of Leptospira spp., the pathogenic bacteria that cause leptospirosis. We examined Leptospira exposure and carriage in livestock on the island of St. Croix, United States Virgin Islands (USVI). We utilized the microscopic agglutination test (MAT) to evaluate the sera, and the fluorescent antibody test (FAT), real time polymerase chain reaction (rt-PCR), and bacterial culture to evaluate urine specimens from livestock (n = 126): 28 cattle, 19 goats, 46 pigs, and 33 sheep. Seropositivity was 37.6% (47/125) with agglutinating antibodies to the following serogroups identified: Australis, Djasiman, Icterohaemorrhagiae, Ballum, Sejroe, Cynopteri, Autumnalis, Hebdomadis, Pomona, Canicola, Grippotyphosa, and Pyrogenes. Urine from 4 animals (4.0%, 4/101) was positive by rt-PCR for lipL32: 2 sheep, 1 goat, and 1 bull. Sequencing of secY amplicons identified L. interrogans in 1 sheep and 1 bull. Livestock in USVI harbor pathogenic Leptospira bacteria and could play a role in the zoonotic cycle of leptospirosis.

Comparison of whole genome sequencing to restriction endonuclease analysis and gel diffusion precipitin-based serotyping of Pasteurella multocida.

The gel diffusion precipitin test (GDPT) and restriction endonuclease analysis (REA) have commonly been used in the serotyping and genotyping of Pasteurella multocida. Whole genome sequencing (WGS) and single nucleotide polymorphism (SNP) analysis has become the gold standard for other organisms, offering higher resolution than previously available methods. We compared WGS to REA and GDPT on 163 isolates of P. multocida to determine if WGS produced more precise results. The isolates used represented the 16 reference serovars, isolates with REA profiles matching an attenuated fowl cholera vaccine strain, and isolates from 10 different animal species. Isolates originated from across the United States and from Chile. Identical REA profiles clustered together in the phylogenetic tree. REA profiles that differed by only a few bands had fewer SNP differences than REA profiles with more differences, as expected. The GDPT results were diverse but it was common to see a single serovar show up repeatedly within clusters. Several errors were found when examining the REA profiles. WGS was able to confirm these errors and compensate for the subjectivity in analysis of REA. Also, results of WGS and SNP analysis correlated more closely with the epidemiologic data than GDPT. In silico results were also compared to a lipopolysaccharide rapid multiplex PCR test. From the data produced in our study, WGS and SNP analysis was superior to REA and GDPT and highlighted some of the issues with the older tests.