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.


Materials and methods
Sample collection. Field activities and euthanasia procedures were in accordance with CDC IACUC Protocol Numbers 2879SALMULX-A4 and AVMA Guidelines for Euthanasia of Animals 11 , and in compliance with the ARRIVE guidelines. A pilot study was performed in STX at a single unique study site in September 2019 to assess logistics of field sampling and laboratory processing and shipment. In total, 39 Sherman Traps® (H.B. Sherman Traps, Tallahassee, Florida, USA) were deployed, and three Mus musculus were sampled. The cross-sectional field study was carried out during June 15-June 30, 2020 and employed single sample events at 20 different study sites from three islands as follows: eight in STX, six in STT and six in STJ (Table 1 and Fig. 1). Trapping consisted of ten 6 × 6 × 18-inch (15 × 15 × 46 cm) Tomahawk® live traps (Tomahawk Live Trap Co, Tomahawk, Wisconsin, USA) and 80 Sherman Traps®, 15 m apart at each study site. For bait, oat and peanut butter mixture were used in the Sherman Trap®; Vienna sausage was used in Tomahawk traps. Traps were placed in rural areas (e.g., farms, parks, or bush) throughout USVI in the evening and collected at dawn for a single sampling event per site.
Because of finite laboratory resources and field and animal safety in a tropical climate, sampling was limited to a maximum of 10 rodents per field site after capture of 33 rodents in Haypenny Beach during the second day of the study (Table 1). Thereafter, any rodents captured surplus to sampling needs were immediately released back into their environment. To avoid selection bias, if more than 10 rodents were captured at a sampling site, www.nature.com/scientificreports/ traps with captured rodents were randomly selected using a random number generator for sampling. Species was confirmed visually, and selection preferred a 50/50 split between Mus musculus and Rattus rattus, if possible. Rodents were rapidly anesthetized with isoflurane until euthanasia by cervical dislocation. Sex, mass, and morphometrics (e.g., total body length, ear, hind foot, and tail lengths) were recorded. Blood samples were collected by cardiac puncture and stored on ice. Samples were centrifuged (15,000×g for 15 min) and serum collected and stored at −20 °C. Frozen serum was then transported to the National Veterinary Services Laboratories, APHIS, U.S. Department of Agriculture (USDA), Ames, Iowa. One kidney from each rodent was removed using aseptic technique by necropsy and immediately stored in Hornsby-Alt-Nally (HAN) media 12 , then transported by overnight delivery services at ambient temperature to the National Animal Disease Center, ARS, USDA, Ames, Iowa. Kidney sample processing. The kidney was macerated in 9 mL of HAN media in 710 mL Whirl-Pak® bags (Nasco).

Microscopic agglutination test (MAT
Dark-field microscopy (DFM). Ten microliters of the macerate were placed on a microscope slide and cover-slipped. Ten fields were examined by DFM (× 200 and × 400) for leptospires.
Culture. One mL of the kidney macerate was used to inoculate 9 mL HAN liquid medium and 200 µl of this dilution was inoculated into 5 mL of three different media: semisolid T80/40/LH 14 that was incubated at 29℃, and 5 mL of liquid and semisolid HAN, which were incubated at 37℃ in 3% CO 2 12 . Semisolid cultures were observed using a lighted black background to examine for development of a Dinger's zone (DZ), and if noted, were confirmed as positive by DFM, at days 3 and 5, weekly for one month, and monthly thereafter for six www.nature.com/scientificreports/ months. From the positive cultures, average time for a DZ to appear was noted. Inoculated tubes of liquid HAN were examined daily by DFM for leptospires from day 3 to day 8.

Fluorescent Antibody Testing (FAT).
A 10 µl aliquot of the kidney macerate was placed on a glass slide within a 7 mm well, in duplicate, and FAT performed as previously described 15 .
DNA extraction. DNA was extracted from 500 μL of kidney macerate using the Maxwell RSC Purefood Purification Pathogen kit (Promega Corporation, Madison, Wisconsin, USA), following manufacturer's instructions, but using 1 h of incubation with lysis buffer A and a 100μL elution volume. For cultures, DNA was extracted from 5 mL of each isolate in HAN media, which was harvested by centrifugation at 10,000×g for 15 min.
Real-time polymerase chain reaction (rtPCR). After DNA extraction from 500 µl of kidney macerate, 5 µl was used for rtPCR. The lipL32 gene was amplified using a set of primers and protocol as described previously: LipL32-47Fd (5′-GCA TTA CMGCT TGT GGTG -3′) and LipL32-301Rd (5′-CCG ATT TCG CCW GTTGG -3′), the probe LipL32-189P ( Table 2). The serogroup for each isolate was assigned according to the antiserum that gave the highest agglutination titer 18 . Genotyping of Leptospira directly from kidney samples. The secY housekeeping gene was amplified with the primers secY_F (5′ -ATG CCG ATC ATT TTT GCT TC-3′) and secY_R (5′-CCG TCC CTT AAT TTT AGA CTT CTT C-3′) 19 . PCR products were then purified and labeled using the Big Dye Terminator v3.1 cycle sequencing reagent (Applied Biosystems, Foster City, California, USA). Sequencing was performed using the ABI 3130XL Genetic Analyzer. Sequence data were analyzed with DNAStar's Lasergene sequence analysis software. Consensus sequences were compared with available sequences in the GenBank database using BLAST. Phylogenetic analyses were performed as described previously. The secY sequence was deposited in NCBI, accession number MZ241295.

Evaluation of virulence.
All animal experimentation was conducted in accordance with protocols as reviewed and approved by the Animal Care & Use Committee at the National Animal Disease Center (ARS-2018-745), and as approved by USDA institutional guidelines. Five representative rodent isolates of L. borgpetersenii (designated LR45, LR47, LR59, LR88, and LR131) were propagated in liquid HAN medium 12 supplemented with 0.4% rabbit serum at 37 °C in 3% CO 2 and evaluated for virulence by intraperitoneal injection into five groups of four golden Syrian hamsters (Mesocricetus auratus), as previously described 15 . One group of four animals was also inoculated through the conjunctival route with 10 8 of strain LR131 in 10 µl of HAN medium, which was applied to the conjunctival membrane of the left eye, as previously described 20 . Liver and kidney tissue were harvested for culture, FAT, and lipL32 rtPCR when hamsters met euthanasia criteria attributable to clinical signs of infection including weight loss, lethargy, bloody discharge from the nose or urogenital tract, and sudden death 21 .

Detection of leptospires in rodent kidney.
A positive carrier status was identified for leptospires in 64 (45.7%) rodents as defined by a positive result in any of the assays used; 57 (89.1%) were positive by culture, FAT and rtPCR; three samples were positive by culture and rtPCR; three samples were positive only by FAT and one sample was positive only by rtPCR. Notably, two rodents identified as carriers were seronegative. All data is presented in Supplementary Table 3.

Evaluation of virulence.
Intraperitoneal inoculation of all hamsters with 10 8 leptospires of each of five strains resulted in acute disease requiring all to be euthanized at 5 days post-infection. Liver and kidney samples from each group were positive by culture, FAT and lipL32 rtPCR. A single group inoculated by the conjunctival route using isolate LR131 showed acute disease by 11 days post-infection; liver and kidney samples from this group also tested positive by culture, FAT and lipL32 rtPCR.

Discussion
Sampling of USVI rodents determined that 45.7% (64/140) were carriers of pathogenic Leptospira species (L. borgpetersenii, L. kirschneri, or L. interrogans) as defined by at least one positive FAT, rtPCR, or culture. Seroprevalence for rodents in this study was relatively high (53.6%; 60/112), compared with previous findings from Central American countries including Barbados (32.6%) 22 , Grenada (24.5%) 23 , Guadeloupe (32%) 22 , and Trinidad (20.5%) 24 . Reactivity to serogroup Ballum was most frequently detected and differs from other studies with rodents in the same region, which reported reactivity primarily to serogroup Icterohaemorrhagiae 9,22-24 . Previous work demonstrated that goats in USVI were also reactive to serogroups Ballum and Icterohaemorrhagiae 8 . However, a positive serology in reservoir hosts of infection is indicative only of exposure and not active disease 4 .
Culture is the definitive diagnostic assay to detect shedding of leptospires, though it can have low sensitivity due to the fastidious nature of Leptospira 4 . The use of the newly described HAN media allowed for recovery of Leptospira species in a significantly shorter time frame at 37℃, compared with T80/40/LH at 29℃, and likely attributable to growth factors and conditions that more closely emulate the in vivo environment to support metabolic requirements of in vivo derived leptospires 12 . Recovery of isolates from rodent hosts allows for their more complete characterization at the genotypic and phenotypic level, and their use for enhanced diagnostic (i.e., animal and human) or bacterin-based vaccination strategies of animals to limit zoonotic transmission.
We attributed 80% of active rodent infections to L. borgpetersenii serogroup Ballum and 5% to L. kirschneri serogroup Icterohaemorrhagiae. One lipL32 rtPCR positive, but culture negative sample was genotyped as L. interrogans directly from kidney 25 . Among nine culture positive samples, species identification was not readily apparent and likely attributable to mixed populations of species. Further analysis of these mixed bacterial samples is underway to obtain clonal isolates, along with a comprehensive analysis of genomes of all recovered isolates. Notably, all mixed and L. kirschneri carriage in rodents were limited to those rodents trapped on St. Croix island. St. Croix rodent populations may have developed a unique carrier population of Leptospira because of its remote location and agricultural landscape.
Identification of L. borgpetersenii, L. kirschneri, and L. interrogans in kidneys of rodents in USVI is similar to recent findings in mongoose from USVI which were carriers of L. borgpetersenii, L. kirschneri and L. interrogans species 26 . However, and in contrast to USVI mongoose which were carriers of L. borgpetersenii serogroup    www.nature.com/scientificreports/ Two species of introduced (non-native) rodents were trapped; Mus musculus and Rattus rattus 39 . The rodent species diversity observed in rural areas in USVI was not as high as that observed in other rural settings in nearby countries 40,41 ; a predominance of Rattus species are frequently noted within the Caribbean, including Grenada, Guadeloupe, Trinidad and Barbados 9,22-24 , which is in contrast to Puerto Rico 37 and results reported here for USVI in which trapped rodents were predominantly M. musculus (80%).
Exposure to rodents is associated with an increased risk of leptospirosis 42 . Serogroup Ballum, the principal reservoir of which is mice 36 , is increasingly reported in human infections 31,43 and our results highlight the need for rodent control to limit effects of leptospirosis 36 . Human leptospirosis infections usually reflect serogroups maintained by local animal populations highlighting the need for serovar-specific vaccine development in highrisk populations. Serogroup Ballum is not included in commercial bacterins for animals despite evidence of infection and its association with poor animal reproductive performance 8,44 .
USVI's climate is typical of maritime tropical environments, with warm and stable temperatures and steady winds. Intense rainfall events generally occur in the form of tropical depressions, storms, or hurricanes. Occurrence of natural disasters and deficiencies in sanitary infrastructure can create a favorable environment for rodent proliferation and increase risk for infection 45 . The first documented case of human leptospirosis was identified in USVI after Hurricanes Irma and Maria, and associated with exposure to flood water and occupation of buildings with evidence of rodent infestation 6 . Research should be conducted in rural and urban USVI areas to identify region-specific risk factors for infection. Leptospirosis is a reemerging disease of public health importance with respect to morbidity and mortality both in humans and animals.
Limitations of this study include using a cross-sectional design that does provide a geographically encompassing assessment of rodents throughout USVI, but in a limited time frame (i.e., two weeks). Rodents can be transient carriers of Leptospira, but we did not control for seasonal variation.
In conclusion, this study confirms the presence of three species of pathogenic Leptospira (L. borgpetersenii, L. kirschneri, and L. interrogans) among USVI's rodent populations. Local isolates of L. borgpetersenii serogroup Ballum and L. kirschneri serogroup Icterohaemorrhagiae should be included in MAT diagnostic panels for human and domestic animal samples from USVI, to increase capacity for disease detection in humans and animals. Control, public health surveillance, and prevention efforts need to be multidisciplinary and multisectoral, making it a prime candidate for the One Health approach. www.nature.com/scientificreports/