# Isolation of pathogenic Leptospira strains from naturally infected cattle in Uruguay reveals high serovar diversity, and uncovers a relevant risk for human leptospirosis

Leticia Zarantonelli, Alejandra Suanes, Paulina Meny, Florencia Buroni, Cecilia Nieves, Ximena Salaberry, Carolina Briano, Natalia Ashfield, Caroline da Silva Silveira, Fernando Dutra, et al.

► To cite this version:

Leticia Zarantonelli, Alejandra Suanes, Paulina Meny, Florencia Buroni, Cecilia Nieves, et al. Isolation of pathogenic Leptospira strains from naturally infected cattle in Uruguay reveals high serovar diversity, and uncovers a relevant risk for human leptospirosis. PLoS Neglected Tropical Diseases, 2018, 12 (9), pp.e0006694. 10.1371/journal.pntd.0006694. pasteur-01881268

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RESEARCH ARTICLE

# Isolation of pathogenic *Leptospira* strains from naturally infected cattle in Uruguay reveals high serovar diversity, and uncovers a relevant risk for human leptospirosis

Leticia Zarantonelli¹,²*, Alejandra Suanes³*, Paulina Meny⁴*, Florencia Buroni⁵*, Cecilia Nieves¹‡, Ximena Salaberry³‡, Carolina Briano⁶‡, Natalia Ashfield⁴, Caroline Da Silva Silveira⁷, Fernando Dutra⁶, Cristina Easton³, Martin Fraga⁷, Federico Giannitti⁷, Camila Hamond²,⁷, Melissa Macías-Rioseco⁷, Clara Menéndez⁴, Alberto Mortola³, Mathieu Picardeau⁸,⁹, Jair Quintero⁴, Cristina Ríos⁴, Víctor Rodríguez⁵, Agustín Romero⁶, Gustavo Varela⁴, Rodolfo Rivero⁵*, Felipe Schelotto⁴*, Franklin Riet-Correa⁷*, Alejandro Buschiazzo¹,⁹,¹⁰*, on behalf of the Grupo de Trabajo Interinstitucional de Leptospirosis Consortium¹

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1 Laboratorio de Microbiología Molecular y Estructural, Institut Pasteur de Montevideo, Montevideo, Uruguay, 2 Unidad Mixta UMPI, Institut Pasteur de Montevideo + Instituto Nacional de Investigación Agropecuaria INIA, Montevideo, Uruguay, 3 Departamento de Bacteriología, División Laboratorios Veterinarios "Miguel C. Rubino" Sede Central, Ministerio de Ganadería, Agricultura y Pesca, Montevideo, Uruguay, 4 Departamento de Bacteriología y Virología, Instituto de Higiene, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay, 5 División Laboratorios Veterinarios "Miguel C. Rubino" Laboratorio Regional Noroeste, Ministerio de Ganadería, Agricultura y Pesca, Paysandú, Uruguay, 6 División Laboratorios Veterinarios "Miguel C. Rubino" Laboratorio Regional Este, Ministerio de Ganadería, Agricultura y Pesca, Treinta y Tres, Uruguay, 7 Instituto Nacional de Investigación Agropecuaria INIA, Estación Experimental La Estanzuela, Colonia, Uruguay, 8 Unité de Biologie des Spirochètes, Institut Pasteur, Paris, France, 9 Joint International Unit « Integrative Microbiology of Zoonotic Agents » IMiZA, Institut Pasteur de Montevideo, Montevideo, Uruguay / Institut Pasteur, Paris, France, 10 Département de Microbiologie, Institut Pasteur, Paris, France

* These authors contributed equally to this work.

‡ These authors also contributed equally to this work

¶ Membership of the Grupo de Trabajo Interinstitucional de Leptospirosis Consortium is provided in S1 Text.

* rrivero@mgap.gub.uy (RR); felipe@higiene.edu.uy (FS); frcorrea@inia.org.uy (FRC); alebus@pasteur.edu.uy (AB)

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OPEN ACCESS

**Citation:** Zarantonelli L, Suanes A, Meny P, Buroni F, Nieves C, Salaberry X, et al. (2018) Isolation of pathogenic *Leptospira* strains from naturally infected cattle in Uruguay reveals high serovar diversity, and uncovers a relevant risk for human leptospirosis. PLoS Negl Trop Dis 12(9): e0006694. https://doi.org/10.1371/journal.pntd.0006694

**Editor:** Jenifer Coburn, Medical College of Wisconsin, UNITED STATES

**Received:** February 8, 2018

**Accepted:** July 16, 2018

**Published:** September 13, 2018

**Copyright:** © 2018 Zarantonelli et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

**Data Availability Statement:** All relevant data are within the paper and its Supporting Information files. Sequence files for secY genes are available from the GenBank database (accession numbers MH325388, MH325389, MH325390, MH325391, MH325392, MH325393, MH325394, MH325395, MH325396, MH325397, MH325398, MH325399, MH325400, MH325401, MH325402, MH325403, MH325404, MH325405, MH325406, MH325407, MH325408, MH325409, MH325410, MH325411,

## Abstract

Leptospirosis is a neglected zoonosis with worldwide distribution. The causative agents are spirochete bacteria of the *Leptospira* genus, displaying huge diversity of serovars, the identity of which is critical for effective diagnosis and vaccination purposes. Among many other mammalian species, *Leptospira* infects cattle, eliciting acute signs in calves, and chronic disease in adult animals often leading to abortions. In South America, and including in Uruguay, beef and dairy export are leading sources of national income. Despite the importance of bovine health, food safety, and bovine-related dissemination of leptospirosis to humans, extremely limited information is available as to the identity of *Leptospira* species and serovars infecting cattle in Uruguay and the South American subcontinent. Here we report a multicentric 3-year study resulting in the isolation and detailed characterization of 40 strains of *Leptospira* spp. obtained from infected cattle. Combined serologic and molecular typing identified these isolates as *L. interrogans* serogroup Pomona serovar Kennewicki (20
MH325412, MH325413, MH325414, MH325415, MH325416, MH325417, MH325418, MH325419, MH325420, MH325421, MH325422, MH325423, MH325424, MH325425, MH325426, MH325427, MH376290, MH376291, MH376292, MH376293). Sequence files for rrS6S genes are available from the GenBank database (accession numbers MH329307, MH329308, MH329309, MH329310, MH329311, MH329312, MH329313, MH329314, MH329315, MH329316, MH329317, MH329318, MH329319, MH329320, MH329321, MH329322, MH329323, MH329324, MH329325, MH329326, MH329327, MH329328, MH329329, MH329330, MH329331, MH329332, MH329333, MH329334, MH329335, MH329336, MH329337, MH329338, MH329339, MH329340, MH329341, MH329342, MH329343, MH329344, MH329345, MH329346, MH374851, MH374852, MH374853, MH374854).

**Funding:** This work was supported by grants from the Agencia Nacional de Investigación e Innovación (ANII Uruguay) #ALI_1_2014_1_4982 (AB), #FSA_1_2013_1_12557 (AB) and #FSA_X_2014_1_105696 (FR-C). Partial support was also obtained through grants from Institut Pasteur Paris (PTR Program) #PTR30-17, Ministerio de Industria, Energía y Minería (MIEM, Industrial Funds program) #2016-8-2-002671, and FOCEM (MERCOSUR Structural Convergence Fund) #COF03/11; and through institutional funds from Instituto Nacional de Investigación Agropecuaria (INIA), Ministerio de Ganadería, Agricultura y Pesca (MGAP), Instituto de Higiene (IH, Udelar University Medical School) and Institut Pasteur de Montevideo (IPMontevideo). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

**Competing interests:** I have read the journal's policy and the authors of this manuscript have the following competing interests: Alejandro Buschiazzo is an Associate Editor of PLoS Neglected Tropical Diseases. Mathieu Picardeau is a Deputy Editor of PLoS Neglected Tropical Diseases.

strains), *L. interrogans* serogroup Canicola serovar Canicola (1 strain), *L. borgpetersenii* serogroup Sejroe serovar Hardjo (10 strains) and *L. noguchii* (9 strains). The latter showed remarkable phenotypic and genetic variability, belonging to 6 distinct serogroups, including 3 that did not react with a large panel of reference serogrouping antisera. Approximately 20% of cattle sampled in the field were found to be shedding pathogenic *Leptospira* in their urine, uncovering a threat for public health that is being largely neglected. The two *L. interrogans* serovars that we isolated from cattle displayed identical genetic signatures to those of human isolates that had previously been obtained from leptospirosis patients. This report of local *Leptospira* strains shall improve diagnostic tools and the understanding of leptospirosis epidemiology in South America. These strains could also be used as new components within bacterin vaccines to protect against the pathogenic *Leptospira* strains that are actually circulating, a direct measure to reduce the risk of human leptospirosis.

## Author summary

Several species of the genus *Leptospira* cause leptospirosis, a disease that is transmitted from animals to humans (zoonosis). Leptospirosis is the most extended zoonosis worldwide, with over a million human cases each year. *Leptospira* spp. infect a broad range of wildlife and domestic animals, including cattle. In several South American countries beef and dairy exports rank among the most important national income sources, explaining why in Uruguay cattle outnumber human population by a factor of 4. Yet, we did not know which *Leptospira* species and serovariants (serovars) circulate among Uruguayan cattle. Current serologic diagnostic methods and whole killed-cell vaccination approaches, critically depend on using the proper serovars, which are hugely variable in *Leptospira* spp. from different regions of the world. Through a multidisciplinary consortium effort, we now report the isolation and typing of 40 strains of pathogenic *Leptospira* spp. An unexpectedly large variation in terms of species and serovars was found. These data are extremely important: 1- to improve diagnostics by updating the available reference antigen panels; 2- to evaluate the efficacy of novel vaccines; and, 3- to implement efficacious bovine vaccination as a means of reducing the incidence of bovine and human leptospirosis.

## Introduction

Leptospirosis is a zoonotic disease of worldwide importance caused by pathogenic spirochetes belonging to the genus *Leptospira* [1]. It affects humans and a broad range of domestic animals and wildlife. In cattle, leptospirosis is an important cause of reproductive failure, including abortions and stillbirths [2]. Infected bovines also constitute an active reservoir for the spread of the zoonotic disease, especially for humans in direct contact with infected animals including veterinarians, abattoir and farm workers, hunters, as well as scientists handling laboratory animals or during fieldwork [3, 4]. Domestic and wild animals are important reservoirs in rural areas, unlike urban settings where rats play a major dissemination role [5, 6]. Human infection with *Leptospira* spp. results from direct exposure if the source of infection is animal tissue, body fluids or urine, and from indirect exposure if the source is environmental, such as soil or urine-contaminated water. While the disease is endemic in many countries, it often presents
screened by the microscopic agglutination test (MAT) [21] for preexisting antibodies against *Leptospira* (S1 Table). Farm selection for subsequent sample collection prioritized those herds with presumptive diagnosis of leptospirosis (MAT titers ≥200 against ≥1 pathogenic *Leptosphaera* reference serogroups). Farms with recorded history of abortions, infertility or acute disease, were also prioritized. Selected farms were visited from January 2015 to September 2017, and individual blood and urine samples from 19 animals were collected (aiming for ≥1 seropositive animal with a 95% confidence interval, using a conservative seroprevalence figure of ≥15% on a reference population of 1000 individuals; seroprevalence estimates from background serologic data in Uruguay are actually higher; the number of individual animals to sample was calculated with the software WinEpi http://www.winepi.net). Due to logistic constraints, in a few cases the number of animals per herd was slightly higher, overall sampling a total of 963 individual animals. Individuals to be sampled in each farm were selected according to recorded history when available, prioritizing animals with clinical signs of acute disease (especially calves with rectal temperature ≥ 39.5°C, jaundice and/or hemoglobinuria), previous antibody titers ≥200 by MAT, and/or history of abortions or infertility. If less than 19 animals met the latter criteria, additional animals (heifers or adult cows) from the same herd were included to complete the required number. A questionnaire was distributed to farmers, gathering information about history of leptospirosis and recent vaccination (<12 months) in the farm.

Blood samples were collected by coccygeal venipuncture using 5 mL tubes with clot activator. Sera were then stored at -20°C. Intramuscular administration of diuretics (~150 mg furosemide, Furo R, Ripoll) and thorough genital organ cleansing (wiping with 70% ethanol) preceded urine collection from individual animals. Approximately 60 mL of midstream urine was collected in sterile 120 mL containers (Bioset, Medicplast).

Urine samples (100 µL) were inoculated in the field, immediately or within 2 h of sample collection (for the rationale, see first section of Results), in 5 mL Ellinghausen-McCullough-Johnson-Harris (EMJH) medium (prepared with Leptospira Medium Base EMJH [Gibco] and albumin BovoLep [Bovogen Biologicals PTY Ltd]), supplemented with 100 µg/mL 5-fluorouracil (5-FU; Sigma) [21], and transported at 4°C to the laboratory together with the corresponding blood/serum samples in Vacutainer tubes (Vacutainer, BD-NJ, USA). In the laboratory, two serial 1:50 dilutions were made from the first urine-inoculated tube, in 5 mL EMJH medium supplemented with 5-FU (EMJH/FU), and all three dilutions were incubated at 29°C. The remaining volume of urine samples was conserved at 4°C for subsequent *lipL32* gene amplification (see below). Sera were used to determine anti-*Leptospira* titers by MAT following reported procedures [21]. Routine MAT tests used the national guide of positivity cut-off at titers ≥200. For comparison of reference *vs* local strains as MAT antigens (S5 Table), sera from animals from which pathogenic *Leptospira* spp. were isolated (only from those herds with no recent vaccination history) were tested by serial two-fold dilutions [21] starting from 1:100. The local strains used for the latter MATs, were chosen to represent each of the different serogroups identified in this work (IP1506001, IP1605021, IP1611024, IP1611025, IP1512017, IP1703027, IP1711049 and IP1512011, according to the numbering scheme defined in Table 1).

## Urine and kidney samples from abattoirs

Random samples of urine (vesical puncture) and kidneys were obtained at 22 slaughterhouses that received animals from geographic regions throughout the country. No indications of reproductive failure nor of any other health problems were recorded for slaughtered animals. Due to pipeline logistics at slaughterhouses, kidneys and urine samples did not correspond to
the same animal such that individual samples were treated as independent. Urine samples were immediately inoculated in EMJH/FU, according to the same protocol as with field samples. Kidneys were transported in 4°C-refrigerated boxes to the laboratory and processed on arrival, 2-6 hours after sampling. A fragment of approximately 10 g of tissue was placed in a funnel, surface-sterilized by dousing with alcohol and flamed with a Bunsen burner. The tissue was then placed in a sterile stomacher bag and 10 mL of phosphate-buffered saline (PBS) were aseptically added. After breaking the tissue down to a pulp in the stomacher machine, the obtained suspension was allowed to settle for 15 minutes, 250 µL of supernatant were drawn and inoculated in 5 mL EMJH/FU (called tube A). From tube A, 500 µL were transferred to a second 5 mL EMJH/FU tube (tube B), thus obtaining also a 10-fold diluted culture. Finally, a third culture was also prepared from each sample by directly inoculating 5 mL Fletcher medium with a small cylinder of kidney tissue obtained with a Pasteur pipette. All cultures were incubated at 29°C.

## Culture conditions, isolation and conservation of *Leptospira* strains

In order to define a precise protocol for culture inoculation in the field after urine collection, decreasing numbers of *L. borgpetersenii* serovar Hardjo strain Sponselee cells, ranging from $10^7$ to 1 bacterium, were incubated in 1 mL filter-sterilized bovine urine. After variable times, 100 µL urine were inoculated in 5 mL EMJH for culture, and bacterial growth weekly monitored under a dark-field microscope.

For isolations, *Leptospira* cultures were incubated at 29°C and observed under dark-field microscopy weekly for up to 6 months [21]. In case of contamination by other microorganisms, the cultures were filtrated through a 0.22 µm sterile syringe filter (Millipore Corporation, MA, USA) and sub-cultured in fresh EMJH media. As soon as spirochete-like bacteria grew in specific cultures, the presence of pathogenic *Leptospira* species was assessed by PCR amplification of the *lipL32* gene (see below). Once no contamination observed, PCR-confirmed cultures were sub-cultured in EMJH media without 5-FU until exponential growth phase. *Leptospira* spp. isolates were then conserved at $\ge 10^8$ cells/mL in EMJH with 2.5% of dimethyl sulfoxide (Sigma) and flash-cooled in liquid nitrogen.

## *lipL32* PCR in urine samples and positive cultures for *Leptospira*

The *lipL32* gene was chosen as a marker of pathogenic *Leptospira* species [22–24]. PCR amplification of *lipL32* was performed using purified DNA from 10 mL of bovine urine samples. The urine was centrifuged at 10,000 g for 15 min, the pellet rinsed once with PBS pH 7.4, and total DNA was extracted with the PureLink Genomic DNA MiniKit (Invitrogen). *lipL32* PCR-amplification was achieved using oligonucleotide primers *lipL32F* (5´-ATCTCCGTTGCACT CTTTGC-3´) and *lipL32R* (5´-ACCATCATCATCATCGTCCA-3´) [25]. The PCR was performed in 50 µL 10 mM Tris.HCl pH 8.4, 50 mM KCl, 1.5 mM MgCl₂, 200 µM dNTPs, 0.25 mg/mL bovine serum albumin (Sigma), 2 µM oligonucleotide primers, 1 U Taq DNA polymerase (Invitrogen) and 5 µL template DNA. PCR cycling comprised 1 denaturation step (5 min at 95°C), 35 amplification cycles (each cycle 30 s at 94°C, 30 s at 58°C and 1 min at 72°C) and a final extension step (7 min at 72°C). PCR products were analyzed by agarose gel electrophoresis and ethidium bromide staining, seeking for the expected 474 bp amplicon. Bovine serum albumin (Sigma) was added in the PCR reaction mix, 0.25 mg/mL, greatly reducing sporadic inhibitory effects of certain urine samples on the amplification reaction. An internal control was always included to quantify this potential inhibition issue, by spiking analyzed samples with 40 ng of *L. borgpetersenii* DNA. Positive amplifications products were randomly chosen in a few field samples, and sequenced confirming specific amplification of *Leptospira* DNA.
This *lipL32* PCR procedure was also performed to rank bacterial cultures (prioritizing more careful follow-ups), after DNA purification from 1 mL of EMJH cultures where suspect spirochetes had been observed by dark-field microscopy.

## Determination of *Leptospira* species by PCR amplification and partial sequencing of the 16S ribosomal RNA gene

DNA from *Leptospira* spp. bovine and human isolates were purified from 1 mL of EMJH culture using the PureLink Genomic DNA MiniKit (Invitrogen). Primers *LeptoA* (5'-GGCGGC GCGTCTTAAACATG-3') and *LeptoB* (5'-TTCCCCCATTGAGCAAGATT-3') were used to amplify the 5'-terminal 331 bp fragment of the 16S rRNA gene (*rrs*) as previously described [26]. The resulting amplicons were sequenced in both senses using internal primers *LeptoC* (Forward) (5'-CAAGTCAAGCGGAGTAGCA-3') and *Rs4* (Reverse)(5'-TCTTAACTGCTG CCTCCCGT-3'). Sequence quality was verified with the Chromas software, and consensus sequences were defined using BioEdit. All *rrs* sequences were deposited in GenBank (S2 Table). Consensus sequences were then compared with available sequences in GenBank using BLAST.

## Multilocus variable-number tandem repeat analysis

Multilocus variable-number tandem repeat (VNTR) analyses were performed according to published methods [27] using five discriminatory markers for VNTR loci 4, 7, 10, Lb4 and Lb5. Purified DNA from each isolate was used to amplify the VNTR4, VNTR7 and VNTR10 loci in *L. interrogans*, and the VNTR10, VNTRLb4 and VNTRLb5 loci in *L. borgpetersenii*. The GelAnalyzer 2010a software (http://www.gelanalyzer.com) was used to analyze the ethidium bromide-stained agarose electrophoresis gels, in which PCR products were resolved in parallel to 100-bp DNA ladder (Thermo Scientific) as molecular weight marker. The number of repeats for each VNTR locus was determined as: number of repeats = [PCR product size(bp)—flanking region (bp)] / repeat unit length (bp).

## Partial *secY* gene sequencing and analysis

DNA from *Leptospira* spp. bovine and human isolates were purified from 1 mL of EMJH culture using the PureLink Genomic DNA MiniKit (Invitrogen). The *secY* gene was partially amplified by PCR with primers *SecYF* (5'-ATGCCGATCATTTTTGCTTC-3') and *SecYR* (5'-CCGTCCCTTAATTTTAGACTTCTTC-3') as described [28]. The resulting 549 bp amplicon was sequenced in both senses. Sequence quality was verified with the Chromas software, and consensus sequences were defined using BioEdit. All *secY* sequences were deposited in GenBank (S2 Table) and compared to those available in PubMed, MLST (https://pubmlst.org/leptospira) and PATRIC (https://www.patricbrc.org) [29] databases. The phylogenetic analyses based on *secY* sequences were performed with MEGA 6.0 software (www.megasoftware.net) using the neighbor-joining method. The evolutionary distances were computed using the Tamura-Nei method and are in the units of the number of base substitutions per site. The reliability of branches was validated by generating 1000 bootstrap replicates. Based on the analysis of sequence similarities, *secY* genotypes were assigned.

## Serotyping

To determine the serogroup of isolated *Leptospira* strains, MAT was used with a panel of serogroup-specific rabbit antisera, spanning 24 *Leptospira* serogroups (KIT Royal Tropical Institute, S3 Table), performed in microtiter plates, mixing equal volumes of viable leptospires with
serial 2-fold dilutions of each rabbit antiserum. After 2 h incubation at 37°C, agglutination of
bacteria was observed under dark-field microscopy. The strain's serogroup was assigned
according to the antiserum that gave highest agglutination titer. Based on the combination of
results from both serogroup determination and molecular typing (rrs gene partial sequencing
and VNTR analysis), a presumptive serovar was assigned to all isolates belonging to L. interro-
gans, and L. borgpetersenii species, as previously described [27].

**Results**

Bovine urine affects *Leptospira* viability

Initial attempts to isolate *Leptospira* strains from bovine urine samples were unsuccessful. The initial protocol was based on collecting the urine from all sampled animals, and then inoculating them into the tubes with culture media. We asked whether bacterial cell viability could be compromised due to exposure to urine over time. As a first approach to address this issue, the particularly fastidious *L. borgpetersenii* serovar Hardjo was chosen [30] to perform *in vitro* tests of viability kinetics in bovine urine. Indeed, a critical maximum time of exposure was defined at less than 2 h (S4 Table), above which subsequent isolation success rates decreased significantly. Although it cannot be ruled out that other serovars might behave differently, based on these observations, all urine samples were inoculated in the field within 2 h of collection, resulting in successful isolations.

PCR screening of urine samples is key to prioritize culture follow-ups
toward isolation

A second logistic challenge for isolation efforts from urine samples, was the high number of
cultures subject to follow-up under dark-field microscopy. PCR amplification of *Leptospira
lipL32* gene was optimized on bovine urine, eventually resulting in a robust method to priori-
tize cultures (Fig 1), identifying those samples that proved positive for pathogenic *Leptospira
spp*. A strong inhibitory effect on *lipL32* PCR amplification was frequently observed, depen-
dent on the urine sample (Fig 1A). This sample-dependent inhibition issue was solved by
washing the bacterial pellet obtained after urine centrifugation with PBS pH 7.4 (Fig 1B), and
then adding bovine serum albumin in the PCR mix (Fig 1C). The sensitivity of this PCR
method was ≥100 *Leptospira* cells, estimated by spiking known amounts of bacteria to sterile
urine samples. Specificity was assessed confirming a positive reaction with relevant serovars of
pathogenic *Leptospira* species (*L. interrogans*, *L. noguchii*, *L. weilii*, *L. borgpetersenii* and *L. san-
tarosai*), while undetectable with non-pathogenic *Leptospira* (*L. biflexa*) nor with unrelated
species (*Escherichia coli*, *Pseudomonas aeruginosa*, *Salmonella* sp., *Staphylococcus aureus* and
*Enterococcus* sp.).

Using this screening strategy, the presence of pathogenic *Leptospira* spp. DNA was con-
firmed in 193 urine samples, indicating that at least ~20% (193/963) of all studied animals
were excreting pathogenic *Leptospira* in their urine (Fig 1D and 1E). False positive results
from collected samples are highly unlikely, considering that *lipL32* is only present in the
genomes of pathogenic *Leptospira* species [22], that no detectable amplification was observed
with non-specific bacteria, and that randomly chosen amplicons from bovine urine samples
confirmed 100% sequence identity with *Leptospira lipL32*. An environmental source of patho-
genic bacteria during urine sample collection is highly unlikely as well, considering the sample
collection procedure and the number of bacteria needed to attain the PCR sensitivity thresh-
old. Following up with this approach at the herd level, 77% of the farms (37/48) that were stud-
ied, harbored ≥1 animal(s) excreting pathogenic *Leptospira*.
<figure><img src="image_5.png" /><figcaption>**Fig 1.** Screening of pathogenic *Leptospira* spp. in urine samples by PCR amplification of the *lipL32* gene. (A) PCR amplification of the *lipL32* gene, showing on the left side products obtained from 10 mL of urine without previous washing of the pellet, and on the right side the inhibition controls using pure DNA spiking. (B) Same as (A), except that the urine pellets on the left side were previously washed with PBS pH 7.4. (C) Same as (A) and (B), except that on the left side of the ladder urine pellets were previously washed with PBS pH 7.4 and BSA was included in the PCR mix. Asterisks show PCR reactions with total inhibition. (D) Typical *lipL32* amplification results, using optimized conditions as in (C), from randomly chosen urine samples collected in the field. (E) Corresponding inhibition controls for panel (D).</figcaption></figure>

https://doi.org/10.1371/journal.pntd.0006694.g001

## Isolation of native strains of pathogenic *Leptospira* spp. infecting cattle

The sampling strategies, as detailed in Methods, were chosen to maximize the odds of isolating local strains of pathogenic *Leptospira* spp. from infected cattle. A two-pronged approach was followed: i- active and directed sampling in the field, at farms with suspicion of *Leptospira* infection; and, ii- random postmortem sampling of animals at slaughterhouses.
*Field sampling.* A total of 48 farms representing both beef and dairy cattle herds were visited from January 2015 to September 2017. They were distributed in 12 out of the 19 geographic departments in which the Uruguayan territory is divided. A total of 963 urine samples were collected and subjected to bacterial culture attempts and *lipL32* PCR screening. On average, *Leptospira* growth was detected by dark-field microscopy on cultures after 28 days (range 7–56 days).

Cultures that showed suspect bacteria, were subjected to *lipL32* PCR amplification, initially identifying 42 positive cultures from independent urine samples. Considering that 193 urine samples were positive by PCR screening, an estimated recovery rate of 21.7% (42/193) positive cultures from urine samples was achieved. From the original 42 positives, we ultimately obtained 32 pure cultures of *Leptospira* spp. (Table 1) from field animals, representing a 76.2% rate of success in isolating these bacteria from positive cultures, and a 3.3% global isolation success rate when considering the whole set of input urine samples (32/963). This latter figure should not be taken as a prevalence estimation of animals shedding leptospires (PCR-positive urine samples is a better indicator), since challenges in cultivating these fastidious bacteria are included in the global isolation rate.

*Sampling at abattoirs.* A total of 288 kidneys and 289 urine samples (representing 577 individual animals) were collected at slaughterhouses. According to the origin of slaughtered animals, all 19 departments of the country were included. 18 positive cultures of *Leptospira* were identified by dark-field microscopy and PCR amplification (*rrs* and *lipL32* genes), from which 8 isolates were eventually obtained, 3 from urine and 5 from kidney samples (Table 1).

## Identification of autochthonous pathogenic *Leptospira* strains

Overall, a total of 40 strains of pathogenic *Leptospira* were isolated from cattle along the course of this study, and characterized by combining serologic and molecular methods (Table 1). Recalling that initially 60 cultures had proved positive for *Leptospira* growth, the figures reveal that 20 could not be isolated (10 from field animals and 10 from slaughterhouses), due to over-growth by contaminant species. Among the 40 characterized strains, 32 were isolated from live animals in the field (30 from cows or heifers, and 2 from calves with signs of acute leptospirosis), and 8 from adult carcasses at abattoirs (Table 1).

The *Leptospira* species were determined by PCR amplification and partial sequencing of the 16S rRNA gene (*rrs*). Three different pathogenic species were thus identified (Table 1): *L. interrogans* (n = 21), *L. borgpetersenii* (n = 10) and *L. noguchii* (n = 9).

Serogrouping of isolates was performed by MAT with a collection of 24 rabbit antisera against reference pathogenic serovars. All but one of the *L. interrogans* isolates corresponded to serogroup Pomona, the different one belonging to serogroup Canicola. The *L. borgpetersenii* strains all classed within serogroup Sejroe. In contrast, the *L. noguchii* isolates showed a broader variety of serogroups, including Pyrogenes (n = 1), Australis (n = 1), Autumnalis (n = 4), and 3 *L. noguchii* isolates that did not agglutinate with any of the reference antisera used.

Taking into account the identification of species and serogroup, together with the VNTR profiles (S1 Fig), it was possible to assign 20 *L. interrogans* strains to serovar Kennewicki, 1 *L. interrogans* to serovar Canicola, and the 10 *L. borgpetersenii* isolates to serovar Hardjo (Table 1). The serovars of the *L. noguchii* isolates could not be predicted, given that current VNTR profiling tables do not allow yet for serovar assignment of this species.

Twelve *L. interrogans*, five *L. borgpetersenii* and one *L. noguchii* strains, were isolated from farms with no history of vaccination (Table 2). Among such animals, MAT agglutination titers against reference strains were positive in ten cases (considering that national guidelines
<table><caption>Table 2. MAT seroreactivity against reference *Leptospira* antigens and history of vaccination in cattle with positive culture of pathogenic *Leptospira* spp.</caption><thead><tr><th>Strain #</th><th>Species identification</th><th>Serogroup / presumptive Serovar identification</th><th>Seroreactivity of the animal from which the isolate was obtained (serogroup/titer)</th><th>Seroreactivity of other animals in the same herd* (serogroup)</th><th>History of vaccination in the farm</th><th>Antigens included in the vaccine</th></tr></thead><tbody><tr><td>IP1507003</td><td>L. interrogans</td><td>Pomona Kennewicki</td><td>Pomona / 200</td><td></td><td>No</td><td></td></tr><tr><td>IP1509008</td><td>L. interrogans</td><td>Pomona Kennewicki</td><td>nr</td><td>Pomona</td><td>No</td><td></td></tr><tr><td>IP1509009</td><td>L. interrogans</td><td>Pomona Kennewicki</td><td>Pomona / 400</td><td></td><td>No</td><td></td></tr><tr><td>IP1509010</td><td>L. interrogans</td><td>Pomona Kennewicki</td><td>Pomona / 400</td><td></td><td>No</td><td></td></tr><tr><td>IP1512011</td><td>L. interrogans</td><td>Pomona Kennewicki</td><td>nr</td><td>Pomona</td><td>No</td><td></td></tr><tr><td>IP1512014</td><td>L. interrogans</td><td>Pomona Kennewicki</td><td>Pomona / 400</td><td></td><td>No</td><td></td></tr><tr><td>IP1512015</td><td>L. interrogans</td><td>Pomona Kennewicki</td><td>Pomona / 6400</td><td></td><td>Yes (19 dpv)</td><td>L. interrogans serovars Pomona, Hardjo, Grippotyphosa, Icterohaemorrhagiae and Canicola</td></tr><tr><td>IP1512016</td><td>L. interrogans</td><td>Pomona Kennewicki</td><td>Pomona / 800</td><td></td><td>Yes (19 dpv)</td><td>L. interrogans serovars Pomona, Hardjo, Grippotyphosa, Icterohaemorrhagiae and Canicola</td></tr><tr><td>IP1603018</td><td>L. interrogans</td><td>Pomona Kennewicki</td><td>Pomona / 3200</td><td></td><td>Yes (19 dpv)</td><td>L. interrogans serovars Pomona, Hardjo, Grippotyphosa, Icterohaemorrhagiae and Canicola</td></tr><tr><td>IP1609022</td><td>L. interrogans</td><td>Pomona Kennewicki</td><td>Pomona / 1600</td><td></td><td>Yes (19 dpv)</td><td>L. interrogans serovars Pomona, Hardjo, Grippotyphosa, Icterohaemorrhagiae and Canicola</td></tr><tr><td>IP1611026</td><td>L. interrogans</td><td>Pomona Kennewicki</td><td>Pomona / 6400 Sejroe Hardjoprajitno / 1600 Sejroe Hardjoprajitno / 1600 Sejroe Wolffii / 800</td><td></td><td>Yes (26 dpv)</td><td>L. interrogans serovars Icterohaemorragiae, Pomona, Canicola, Wolffii, Hardjo, Tarassovi and Grippotyphosa L. borgpetersenii serovar Hardjo</td></tr><tr><td>IP1710039</td><td>L. interrogans</td><td>Pomona Kennewicki</td><td>Pomona / 6400</td><td></td><td>No</td><td></td></tr><tr><td>IP1710040</td><td>L. interrogans</td><td>Pomona Kennewicki</td><td>Pomona / 6400 Sejroe Hardjoprajitno / 3200 Sejroe Hardjoprajitno / 1600 Sejroe Wolffii / 1600</td><td></td><td>No</td><td></td></tr><tr><td>IP1710043</td><td>L. interrogans</td><td>Pomona Kennewicki</td><td>Pomona / 3200 Sejroe Hardjoprajitno / 800</td><td></td><td>No</td><td></td></tr><tr><td>IP1710044</td><td>L. interrogans</td><td>Pomona Kennewicki</td><td>Pomona / 3200 Serjoe Hardjoprajitno / 3200 Serjoe Hardjoprajitno / 800</td><td></td><td>No</td><td></td></tr><tr><td>IP1710045</td><td>L. interrogans</td><td>Pomona Kennewicki</td><td>Pomona / 6400</td><td></td><td>No</td><td></td></tr><tr><td>IP1710047</td><td>L. interrogans</td><td>Pomona Kennewicki</td><td>nr</td><td>Sejroe Hardjoprajitno Sejroe Wolffii</td><td>No</td><td></td></tr><tr><td>IP1506001</td><td>L. borgpetersenii</td><td>Sejroe Hardjo</td><td>Pomona / 400</td><td></td><td>No</td><td></td></tr><tr><td>IP1509005</td><td>L. borgpetersenii</td><td>Sejroe Hardjo</td><td>nr</td><td>Sejroe Hardjoprajitno Sejroe Wolffii Pomona</td><td>No</td><td></td></tr></tbody></table>

(Continued)
<table><caption>**Table 2. (Continued)**</caption><thead><tr><th>Strain #</th><th>Species identification</th><th>Serogroup / presumptive Serovar identification</th><th>Seroreactivity of the animal from which the isolate was obtained (serogroup/titer)</th><th>Seroreactivity of other animals in the same herd* (serogroup)</th><th>History of vaccination in the farm</th><th>Antigens included in the vaccine</th></tr></thead><tbody><tr><td>IP1509006</td><td>L. borgpetersenii</td><td>Sejroe Hardjo</td><td>nr</td><td>Sejroe Hardjobovis<br>Sejroe Hardjoprjitno<br>Sejroe Wolffii<br>Pomona</td><td>No</td><td></td></tr><tr><td>IP1512013</td><td>L. borgpetersenii</td><td>Sejroe Hardjo</td><td>nr</td><td>Sejroe Hardjobovis<br>Sejroe Hardjoprjitno<br>Sejroe Wolffii<br>Pomona</td><td>No</td><td>na</td></tr><tr><td>IP1605020</td><td>L. borgpetersenii</td><td>Sejroe Hardjo</td><td>Sejroe Wolffii / 200</td><td></td><td>Y (120 dpv)</td><td>L. interrogans serovars Pomona, Hardjo, Grippotyphosa, Icterohaemorrhagiae and Canicola</td></tr><tr><td>IP1704030</td><td>L. borgpetersenii</td><td>Sejroe Hardjo</td><td>nr</td><td>Sejroe Hardjobovis,<br>Sejroe Wolffii</td><td>Yes (nda)</td><td>nda</td></tr><tr><td>IP1704031</td><td>L. borgpetersenii</td><td>Sejroe Hardjo</td><td>nd</td><td>Sejroe Hardjobovis,<br>Sejroe Wolffii</td><td>Yes (nda)</td><td>nda</td></tr><tr><td>IP1708034</td><td>L. borgpetersenii</td><td>Sejroe Hardjo</td><td>nr</td><td>Sejroe Hardjobovis<br>Sejroe Hardjoprjitno<br>Sejroe Wolffii</td><td>No</td><td></td></tr><tr><td>IP1512017</td><td>L. noguchii</td><td>No agglutination§ / na¶</td><td>nr</td><td>nd</td><td>na</td><td></td></tr><tr><td>IP1605021</td><td>L. noguchii</td><td>Pyrogenes</td><td>nr</td><td>Sejroe Hardjobovis<br>Sejroe Hardjoprjitno<br>Sejroe Wolffii</td><td>No</td><td></td></tr><tr><td>IP1611024</td><td>L. noguchii</td><td>Australis / na¶</td><td>nr</td><td>Sejroe Hardjobovis<br>Sejroe Hardjoprjitno<br>Sejroe Wolffii<br>Pomona</td><td>Yes (100 dpv)</td><td>L. interrogans serovars Icterohaemorragiae, Pomona, Canicola, Wolffii, Hardjo, Tarassovi and Grippotyphosa<br>L. borgpetersenii serovar Hardjo</td></tr><tr><td>IP1611025</td><td>L. noguchii</td><td>Autumnalis / na¶</td><td>Sejroe Hardjobovis / 3200<br>Sejroe Hardjoprjitno /<br>3200 Sejroe Wolffii / 1600</td><td></td><td>Yes (26dpv)</td><td>L. interrogans serovars Icterohaemorragiae, Pomona, Canicola, Wolffii, Hardjo, Tarassovi and Grippotyphosa<br>L. borgpetersenii serovar Hardjo</td></tr><tr><td>IP1705032</td><td>L. noguchii</td><td>Autumnalis / na¶</td><td>nr</td><td>Sejroe Hardjobovis<br>Sejroe Wolffii<br>Pomona</td><td>Yes (nda)</td><td>L. interrogans serovar Pomona</td></tr><tr><td>IP1712055</td><td>L. noguchii</td><td>No agglutination§ / na¶</td><td>nr</td><td>Pomona</td><td>Yes (150dpv)</td><td>L. interrogans serovars Icterohaemorragiae, Pomona, Canicola, Wolffii, Hardjo, Tarassovi and Grippotyphosa<br>L. borgpetersenii serovar Hardjo</td></tr></tbody></table>

currently define less than 200 as non-reactive). However, when local isolates were added to the panel of MAT antigens for comparative purposes, 16 out of the 18 sera from non-vaccinated herds showed anti-*Leptospira* titers against the homologous autochthonous strain that was isolated (S5 Table). These results suggest that including local isolates of *Leptospira* spp. in the panel of antigens used for MAT may improve the sensitivity of the method. All the isolates recovered from herds with no history of vaccination, belonged to the homologous serogroup as shown by the seroreactivity data (S5 Table).

<footnote>*Shown if the seroreactivity MAT titer <200 in the animal from which the isolate was recovered</footnote>

<footnote>§ No agglutination against the reference panel of serogrouping antisera</footnote>

<footnote>¶ No molecular proxy available for *L. noguchii* serovar assignment; **na**: not applicable; **nr**: non-reactive (below cutoff MAT titer 200); **nd**: not done; **nda**: no data</footnote>

<footnote>available; **dpv**: days post vaccination when both urine and sera samples were collected</footnote>
## Phylogeny of *Leptospira* isolates based on *secY* gene sequence analysis

Genetic analysis of the 501bp *secY* allele was performed on the 40 typed isolates described in this work. Comparison to other *L. interrogans* (serovars Pomona and Canicola), *L. borgpetersenii* (serovar Hardjo) and *L. noguchii* sequences, obtained from other geographical regions and available in public databases, allowed to build a picture of related groups. Also included in this analysis were *secY* sequences obtained from 4 *Leptospira* strains recently isolated from human infections in Uruguay by one of the groups of our consortium [12, 13]. Such human isolates correspond to *L. interrogans*, *L. kirschneri* and *L. borgpetersenii* species. The dendrogram of partial *secY* sequence clustering, uncovered four phylogenetic clades that corresponded to gen-omospecies identified by partial *rrs* gene sequencing: *L. interrogans*, *L. borgpetersenii*, *L. kirsch-neri* and *L. noguchii* (Fig 2). The same 4-clades scenario emerged by calculating phylogeny with *rrs* gene sequences (S2 Fig). Only one homogeneous cluster was observed for the *L. inter-rogans* *secY* sequences, indicating that bovine isolates from Uruguay belonging to this species have close homology with isolates from South America (mainly from Brazil and Argentina) [31]. It is worth noting that two *L. interrogans* strains that had recently been isolated from human leptospirosis cases in Uruguay affecting rural workers [12, 13] clustered in the same *secY* clade together with the *L. interrogans* bovine isolates that we now describe. Concerning the *L. borgpetersenii* bovine strains, they also clustered with *L. borgpetersenii* serogroup Sejroe isolates from human and bovine sources in South America, Australia and USA; however, they showed no homology with the uruguayan *L. borgpetersenii* human isolate, which belongs to serogroup Ballum (F Schelotto, personal communication). Contrasting with such homoge-neous clustering of *L. interrogans* and *L. borgpetersenii* strains, *secY* sequence analysis of the *L. noguchii* isolates revealed a substantially broader diversity, with isolates grouped in two distinct clusters. The first included two isolates, from Panama and Peru. The second cluster, with slight heterogeneity within, comprised all the *L. noguchii* isolates we are now reporting from Uru-guay, as well as a number of other strains obtained from both human and animal origin in sev-eral countries of the American continent (Brazil, Nicaragua, Peru, Trinidad & Tobago, USA). Worth highlighting, the *secY* sequences of our bovine isolates IP1611024, IP1708035 and IP1709037, are identical to some of the *L. noguchii* strains recently reported in Brazil, isolated from cattle [32] and humans [33].

## Discussion

We are now reporting the isolation and typing of 40 native strains of pathogenic *Leptospira* spp. from infected cattle in Uruguay. This is the first systematic effort to isolate and type autochthonous *Leptospira* strains from cattle in this country, where bovine leptospirosis is a major concern as a cause of abortions and zoonotic dissemination. *L. interrogans* serovar Ken-newicki (serogroup Pomona), our most frequent bovine isolate, has actually been also recov-ered from human patients with leptospirosis in Uruguay [12]. To further confirm this potential link between cattle and humans, we have now shown that the *secY* genotypes of both *L. interrogans* Kennewicki and Canicola serovars, are identical in *Leptospira* strains isolated from patients (rural workers) and from cattle (Fig 2), strongly suggesting that the latter dis-seminate the infection to exposed humans.

The successful culture of leptospires from bovine samples has likely been boosted by opti-mizing field sampling protocols, especially after quantifying time-dependent *Leptospira* viabil-ity in bovine urine. PCR screening has also been instrumental in prioritizing cultures, the number of which increased dramatically due to the systematic use of three culture dilutions per animal, themselves important to improve purity in some cases.
<figure><img src="image_6.png" /></figure>
**Fig 2. Phylogeny of Leptospira spp. isolates based on secY gene sequence analysis.** Evolutionary history inferred by using the Neighbor-Joining method. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Tamura-Nei method and are in the units of the number of base substitutions per site. The analysis involved 76 partial sequences of the secY gene including the 40 bovine isolates from Uruguay that we are now reporting. Uruguayan strains from bovine hosts (in blue) and human patients (green) are compared to 32 additional sequences (in red) corresponding to isolates obtained elsewhere and from a variety of hosts, as indicated within brackets. Asterisks indicate the known serovar for isolates where such information is known, following the code: \* serovar Pomona \*\* serovar Panama \*\*\* serovar Autumnalis \*\*\*\* serovar Hardjo. Isolates obtained in Uruguay are named according to their strain denomination as "IP" (Institut Pasteur Montevideo) or "IH" (Instituto de Higiene) followed respectively by a 7- or a 4-digit number. GenBank accession numbers are indicated in parentheses. Well separated phylogenetic clades have a correspondence to different *Leptospira* species as indicated toward the right of the figure. The Patoc strain at the bottom of the panel belongs to the saprophytic species *L. biflexa*.

https://doi.org/10.1371/journal.pntd.0006694.g002

A total of 963 urine samples that were processed, eventually produced 42 positive cultures. Among these 42, 9 had produced negative PCR results at the time of urine sample screening. Two different scenarios explain such discrepancies: 8 of the 9 negative results, appeared early during our studies, and eventually proved to be the consequence of urine inhibition, triggering the optimization of our protocols (see Methods and Fig 1). Only in one sample we can strongly suggest that it is the PCR method's sensitivity that explains the divergent result. In sum, *lipL32* PCR screening is an instrumental strategy to prioritize culture follow-ups, albeit not leading to discarding ongoing cultures. We are now optimizing a more sensitive real-time PCR approach, anticipated to also being more robust for screening purposes.

Regarding important, and frequently neglected factors that can lead to success or failure in nation-wide efforts based on field sampling, it is worth highlighting the voluntary participation of farmers and private veterinarians. Early arrangements ensuring for such implications were critical logistic factors for a swift sample collection strategy and for gathering useful information about herds and individual animals. Serial dilutions of the biologic samples on separate culture tubes were successfully used as a means to tackle contamination issues. Most of the positive cultures were successfully purified using the first two dilutions A and B, roughly 50% success from each one. Further diluting the inocula (tube C) allowed the recovery/purification of only 4 additional isolates. Overall, EMJH media outperformed Fletcher in our hands, with only two isolates grown from the latter that were also obtained with EMJH.

Combined serologic and molecular approaches revealed the presence of three different *Lep-tospira* species. Besides the anticipated *L. interrogans* and *L. borgpetersenii* species, known to be major infectious agents in cattle [2, 34], an important number of isolates corresponded to *L. noguchii*, both from field samples as well as from abattoirs. *L. noguchii* has been isolated from cattle in South America [14, 32, 33], but had never been reported in Uruguay, and extremely limited information is currently available about its epidemiologic importance. Are *L. noguchii* strains a relevant cause of acute disease or reproductive problems in cattle? One of the two strains that we have isolated from calves with signs of acute leptospirosis, was actually identi-fied as *L. noguchii*, but more information is urgently needed in order to establish the contribu-tion of this unanticipated species in the burden of veterinarian and human leptospirosis in South America. The other strain infecting a suspected acute case was confirmed as *L. interro-gans* serogroup Canicola serovar Canicola, a highly virulent variant often isolated from dogs. Serovar Canicola is however not considered to be adapted to cattle, although it has been reported to infect bovine hosts incidentally, including recent reports in Brazil [35]. It is inter-esting to note that the isolates belonging to *L. interrogans* and *L. borgpetersenii*, displayed lim-ited variation. The latter revealed a single VNTR profile (consistent with a single serovar, Hardjo, within the Sejroe serogroup), also coherent with a unique *secY* genotype (B). As for the *L. interrogans* strains, once again quite homogeneous features were found for all isolates, with 20 out of 21 compatible with serovar Kennewicki (serogroup Pomona), and displaying a single *secY* genotype (A). Only one *L. interrogans* was different, VNTR clearly matching the
one expected for serovar Canicola (in line with Canicola serogroup sero-agglutination), yet sharing the same *secY* genotype A as the Pomona Kennewicki strains. In stark contrast, the 9 *L. noguchii* isolates uncovered an unexpected variety of serogroups. We have not yet assigned serovar types to these *L. noguchii* strains, given that the VNTR multilocus analysis scheme has not been validated for this *Leptospira* species on the basis of cross-agglutinin absorption tests (CAAT) with serovar-specific antisera. We are currently sequencing the whole genomes for all isolates and actively pursuing direct serovar identification by CAAT for the *L. noguchii* strains. However, it can immediately be recognized that all nine *L. noguchii* strains likely correspond to 9 distinct serovars, combining the information of serogrouping and *secY* genotypes. Three of them did not agglutinate with any of the reference antisera tested, which span 24 serogroups that cover major pathogenic *Leptospira* [36]. The other six corresponded to serogroups Pyrogenes, Australis and Autumnalis, the latter including four different isolates, all of which differed in *secY* genotypes (D, F, G and H). The three *L. noguchii* isolates that did not react with serogroup-specific reference antisera, revealed as yet three additional *secY* genotypes (C, F and I), hence likely pertaining to three disparate serovars as well.

Serogroup Pomona is one of the most common variants isolated from animals worldwide [37]. This serogroup displays important genetic diversity, as revealed by restriction endonuclease analysis (REA) [38], even within serovars. However, the REA-based genetic profiles of Pomona serovar Kennewicki, show high stability among isolates from a single outbreak [39] and, interestingly, a strong correlation between specific hosts and corresponding REA profile. Those results are consistent with our study: analyzed by *secY* allele genotyping, a high homogeneity was observed in all Pomona Kennewicki isolates from cattle, despite the broad geographic distribution of the isolates, including those obtained in the field and from slaughterhouses. Serovar Kennewicki is recognized as an animal pathogen [40], apparently adapted to pigs as maintenance host. Even though in Uruguay domestic pigs are not usually raised together with cattle, a forbidden practice in dairy farms, we should not rule out wild boars or other wild animals as potential hosts for this serovar, nor an endemic cycle in domestic cattle [2].

More information is needed to evaluate the prevalence of the serovars we have isolated in the whole country, and neighboring ones in South America. Furthermore, the virulence of these strains in relevant leptospirosis models will be important evidence that must be investigated, regarding pathogenicity (e.g. mortality in the hamster model) and renal colonization (e.g. in the bovine host). It is worth highlighting that we have isolated similar *Leptospira* species and sero-variants from chronic and acute cases in the field, as well as from dead animals from abattoirs, suggesting they represent a genuine sampling of the true population distribution of infectious *Leptospira* spp. in cattle. To be conclusive, an epidemiologic study with national geographic coverage is a necessary next step, as well as an in-depth molecular analysis of the *Leptospira* DNA recovered from PCR-positive urine samples that did not result in positive cultures.

At the individual animal level, and only considering herds with no recent history of vaccination (18 cases), the MAT technique correctly predicted the serogroup (Pomona) of 9 out of the 12 animals where *L. interrogans* strains were isolated (Table 2). In contrast, none of the 5 cases with *L. borgpetersenii* infections, nor the one from which a *L. noguchii* strain was isolated, presented detectable antibody titers using the diagnostic panel of reference available at the national diagnostics laboratory (DILAVE, MGAP). This is likely due to low sensitivity of the MAT, a known issue when it comes to host-acclimated serovars such as Hardjo in cattle [41]. The MAT did not identify any of the *L. noguchii* isolates, as these were not included within the reference antigen panel in the national diagnostics laboratories (DILAVE, Ministry of Livestock, Agriculture and Fishery). This finding is important, as *L. noguchii* is a recognized
pathogenic species for animals and humans [33, 42]. However, when autochthonous *L. interrogans* serogroup Pomona, *L. borgpetersenii* serogroup Sejroe and representative serogroups of the *L. noguchii* strains were included for anti-*Leptospira* antibodies titration by MAT, we did observe an increase of sensitivity: analyzing those herds with no history of recent vaccination, all the animals from which *L. borgpetersenii* strains were isolated showed reactivity against the local isolate, as it was also the case for an animal from which *L. noguchii* serogroup Pyrogenes was isolated (S5 Table).

As a consequence of this study, the inclusion of these native strains among the antigens for MAT diagnostics and seroprevalence epidemiologic studies, must be an immediate action. Such policies will be important to increase MAT-based diagnostics sensitivity and accuracy [43], and to improve the estimations of prevalence and incidence of bovine leptospirosis infection in the country. Furthermore, isolation and characterization of circulating *Leptospira* strains, are ongoing activities as a result of our multicentric consortium efforts. We anticipate that new variants and/or species may be discovered, achieving a more complete understanding of current diversity of *Leptospira* in South America.

A recent study of bovine *Leptospira* spp. isolates obtained from animals in slaughterhouses in Brazil, shows an important diversity in terms of species and serovars [14]. Libonati et al. report two *L. interrogans* strains belonging to serogroup Sejroe, and four different serogroups assigned to each of the other two *L. santarosai* and *L. noguchii* species identified. Our results now demonstrate a similar diversity of bovine isolates in terms of species and serovars. We have isolated *L. borgpetersenii* serogroup Sejroe strains, although so far, no *L. santarosai* isolates nor *L. interrogans* serogroup Sejroe have been recovered. Instead, we did isolate several strains of *L. interrogans* serogroup Pomona (presumptive serovar Kennewicki) and one Canicola (presumptive serovar Canicola). With regards to *L. noguchii*, the broad range of serogroups that we have detected seems to be a shared scenario with the situation in Brazil, with Autumnalis, Australis and Pyrogenes identified in both countries (additionally, serogroup Panama has also been identified in Brazil [32]). However, three *L. noguchii* isolates could not be classified in any serogroup, failing to agglutinate with the broad panel of reference antisera that was used. These results were confirmed in three different laboratories within our consortium, including the Paris center (WHO Collaborating Center and French reference laboratory for leptospirosis). In any case, these novel serogroups are distinct from the *L. noguchii* strains so far isolated in Brazil.

It does not escape our attention that most of the serovars that we are now reporting, are not included in the vaccines currently available to the farmers. Except for *L. borgpetersenii* serovar Hardjo and *L. interrogans* serovar Canicola, to the best of our knowledge neither serovar Kennewicki (*L. interrogans*) nor any of the *L. noguchii* serogroups/serovars that we identified, are being included in bacterin formulations that different companies produce and commercialize as bovine vaccines in South America (Table 2). Bacterins confer little or no cross-protection between serovars, hence the serovars that actually circulate in each region should be included to aim for efficacious vaccines [34]. Indeed, in our study we have obtained several isolates from one herd before and after vaccination. We will now perform closer analyses of naturally exposed herds, following up the effects of vaccination at the individual level. That current vaccines might have shifted the serovar profile of currently circulating *Leptospira* strains in Uruguay, is a plausible scenario. Proper bacterin vaccination should result in herd protection. We should have thus observed lower isolation rates from vaccinated herds, but we have not. Urine shedding of leptospires can be effectively controlled or significantly reduced in livestock, by using the correct bacterin formulations, according to recent studies with naturally exposed sheep herds [44] or with experimental vaccination/challenge approaches in cattle [45]. Significant reduction in bovine renal colonization and bacterial urinary shedding are achieved by
vaccination with bacterins that include the infectious serovars [46], ultimately controlling endemic cycles of infection. Moreover, a systematic vaccination and surveillance program for pig and cattle leptospirosis in New Zealand, demonstrated a correlative dramatic decrease in the incidence, not only of the animal disease, but also of human leptospirosis [47]. Nevertheless, further research is needed to obtain long-lasting vaccination effects and complete protection against bacterial infection. Likely a protective cellular immune response is needed in the cattle model [46, 48, 49] to generate a highly efficacious vaccine against leptospirosis, and not only the humoral response triggered by killed-cell bacterins. The latter are also known to trigger a biased response towards the serovar-specific bacterial lipopolysaccharide antigen, T-independent with lack of memory response [50].

A more thorough understanding of leptospirosis epidemiology, including maintenance hosts and impact in livestock production, is essential to understand and design effective control strategies for this zoonosis. Efficacy studies with currently available vaccines for bovine leptospirosis in our region are also urgently needed. The assembly of this multicentric consortium (S1 Text) gathering the complementary expertise of several key research and governmental institutions in Uruguay, has made possible to obtain the first repository of *Leptospira* isolates in the public domain, most of them already typed in terms of species, serogroup and serovar. This is a major milestone in the way of controlling leptospirosis in Uruguay, with the associated far-reaching aim of reducing the risk for the human population.

**Supporting information**

**S1 Text.** Members of the "Grupo de Trabajo Interinstitucional de Leptospirosis" Consortium.

(DOCX)

**S1 Table.** Reference *Leptospira* strains used as antigens for antibody titration of bovine sera, by microscopic agglutination test.** This panel is defined by the Uruguayan veterinarian health authorities (Ministry of Livestock, Agriculture and Fishery), and used for diagnostic purposes.

(DOCX)

**S2 Table.** GenBank accession numbers for *secY* and *rrs* partial sequences obtained for all the *Leptospira* spp. isolates included in this work.

(DOCX)

**S3 Table.** Reference antisera used for serogroup determination by microscopic agglutination test.

(DOCX)

**S4 Table.** Effect of bovine urine in *L. borgpetersenii* serovar Hardjo cell viability.

(DOCX)

**S5 Table.** MAT of sera from individual animals from which pathogenic *Leptospira* strains were isolated, circumscribed to farms with no history of vaccination (see Table 2). Autochthonous *Leptospira* antigens are compared against the reference panel used by the national health agency.

(DOCX)

**S1 Fig.** Representative profiles of Variable Number of Tandem Repeat (VNTR) analyses of *L. interrogans* and *L. borgpetersenii* autochthonous isolates.** PCR amplifications of VNTR loci 4, 7, 10, Lb4 and Lb5, separated by agarose electrophoresis and ethidium bromide staining.
Representative gels are included corresponding to: *L. interrogans* serogroup Pomona isolates IP1512014 and IP1512016 (lines 1 and 2, respectively); *L. interrogans* serogroup Canicola isolate IP1710049 (line 3) and *L. borgpetersenii* serogroup Sejroe isolates IP1506001, IP170430 and IP1708034 (lines 4, 5, 6, respectively). A PCR negative control is included in each gel, lanes labeled as (-). Molecular weight marker 100bp-ladders are included on side lanes, with a few reference sizes labeled in number of base pairs.
(DOCX)

**S2 Fig. Phylogeny of Leptospira spp. isolates based on rrs sequence analysis.** Dendrogram using the neighbor-joining method (calculated using the Tamura-Nei model) plotting the relatedness of partial sequences of the 16S rRNA gene (rrs) including the 40 bovine isolates from Uruguay (blue labels) that we are now reporting. Sequences from 4 human isolates from Uruguay (green labels) were also included and plotted in comparison to 4 sequences corresponding to reference strains obtained elsewhere (red labels) and from different hosts, as indicated within parentheses. Isolates obtained in Uruguay are named according to their strain denomination as "IP" (Institut Pasteur Montevideo) or "IH" (Instituto de Higiene) followed by a 7- or 4-digit number, and after the vertical bar the GenBank accession number is reported for each one (S2 Table). Well separated phylogenetic clades correspond to different *Leptospira* species as indicated toward the right of the figure. The Patoc strain at the bottom of the panel belongs to the saprophytic species *L. biflexa*, and is included as a phylogenetic distance reference.
(DOCX)

**Acknowledgments**

We thank Julie Collins Emerson and Peter Wilson (Massey University, Palmerston North, New Zealand); Walter Lilenbaum and Ana Paula Loureiro (Universidade Federal Fluminense, Rio de Janeiro, Brazil); and Pascale Bourhy (Institut Pasteur, France), for their helpful suggestions and lively discussions. We also thank Dr. Jorge Mattos (MGAP, Uruguay) for key assistance in obtaining samples at slaughterhouses.

**Author Contributions**

**Conceptualization:** Leticia Zarantonelli, Alejandra Suanes, Rodolfo Rivero, Felipe Schelotto, Franklin Riet-Correa, Alejandro Buschiazzo.

**Data curation:** Leticia Zarantonelli, Alejandra Suanes, Florencia Buroni, Cecilia Nieves.

**Formal analysis:** Leticia Zarantonelli, Alejandra Suanes, Cecilia Nieves, Mathieu Picardeau, Rodolfo Rivero, Felipe Schelotto, Franklin Riet-Correa.

**Funding acquisition:** Rodolfo Rivero, Felipe Schelotto, Franklin Riet-Correa, Alejandro Buschiazzo.

**Investigation:** Leticia Zarantonelli, Alejandra Suanes, Paulina Meny, Florencia Buroni, Cecilia Nieves, Ximena Salaberry, Carolina Briano, Caroline Da Silva Silveira, Martin Fraga, Camila Hamond, Melissa Macías-Rioseco, Gustavo Varela, Rodolfo Rivero, Felipe Schelotto, Franklin Riet-Correa, Alejandro Buschiazzo.

**Methodology:** Leticia Zarantonelli, Alejandra Suanes, Paulina Meny, Florencia Buroni, Cecilia Nieves, Ximena Salaberry, Natalia Ashfield, Cristina Easton, Melissa Macías-Rioseco, Clara Menéndez, Alberto Mortola, Jair Quintero, Cristina Ríos, Víctor Rodríguez, Agustín Romero, Felipe Schelotto, Franklin Riet-Correa.
**Project administration:** Alejandro Buschiazzo.

**Resources:** Paulina Meny, Florencia Buroni, Ximena Salaberry, Fernando Dutra, Cristina Easton, Federico Giannitti, Camila Hamond, Clara Menéndez, Alberto Mortola, Mathieu Picardeau, Franklin Riet-Correa.

**Supervision:** Fernando Dutra, Mathieu Picardeau, Gustavo Varela, Rodolfo Rivero, Felipe Schelotto, Franklin Riet-Correa, Alejandro Buschiazzo.

**Validation:** Leticia Zarantonelli, Alejandra Suanes.

**Writing – original draft:** Leticia Zarantonelli, Franklin Riet-Correa, Alejandro Buschiazzo.

**Writing – review & editing:** Leticia Zarantonelli, Alejandra Suanes, Martin Fraga, Federico Giannitti, Camila Hamond, Mathieu Picardeau, Rodolfo Rivero, Felipe Schelotto, Franklin Riet-Correa, Alejandro Buschiazzo.

## References

1. Picardeau M. Virulence of the zoonotic agent of leptospirosis: still terra incognita? Nat Rev Microbiol. 2017; 15(5):297–307. https://doi.org/10.1038/nrmicro.2017.5 PMID: 28260786

2. Ellis WA. Animal leptospirosis. Curr Top Microbiol Immunol. 2015; 387:99–137. https://doi.org/10.1007/978-3-662-45059-8_6 PMID: 25388134

3. Haake DA, Levett PN. Leptospirosis in humans. Curr Top Microbiol Immunol. 2015; 387:65–97. https://doi.org/10.1007/978-3-662-45059-8_5 PMID: 25388133

4. Benschop J, Collins-Emerson J, Maskill A, O'Connor P, Tunbridge M, Yupiana Y, et al. Leptospirosis in three workers on a dairy farm with unvaccinated cattle. N Z Med J. 2017; 130(1462):102–108. PMID: 28934773

5. Adler B. History of leptospirosis and leptospira. Curr Top Microbiol Immunol. 2015; 387:1–9. https://doi.org/10.1007/978-3-662-45059-8_1 PMID: 25388129

6. Millan J, Cevidanes A, Chirife AD, Candela MG, Leon-Vizcaino L. Risk factors of Leptospira infection in Mediterranean periurban micromammals. Zoonoses Public Health. 2018; 65(1):e79–e85. https://doi.org/10.1111/zph.12411 PMID: 29058382

7. Costa F, Hagan JE, Calcagno J, Kane M, Torgerson P, Martinez-Silveira MS, et al. Global morbidity and mortality of leptospirosis: a systematic review. PLoS Negl Trop Dis. 2015; 9(9):e0003898. https://doi.org/10.1371/journal.pntd.0003898 PMID: 26379143

8. Torgerson PR, Hagan JE, Costa F, Calcagno J, Kane M, Martinez-Silveira MS, et al. Global burden of leptospirosis: estimated in terms of Disability Adjusted Life Years. PLoS Negl Trop Dis. 2015; 9(10): e0004122. https://doi.org/10.1371/journal.pntd.0004122 PMID: 26431366

9. Caffarena RM CR, Cascelli ES, Martínez ES. Avances en leptospirosis en el Uruguay. Rev Urug Pat Clín Microbiol. 1971; 9:186–194.

10. Repiso MV, Gil A, Bañales PM, D'Anatro N, Fernández L, Guarino H, et al. Prevalencia de las principales enfermedades infecciosas que afectan el comportamiento reproductivo en la ganadería de carne y caracterización de los establecimientos de cría del Uruguay. Veterinaria (Montevideo). 2005; 40(157):5–28.

11. Schelotto F, Hernandez E, Gonzalez S, Del Monte A, Ifran S, Flores K, et al. A ten-year follow-up of human leptospirosis in Uruguay: an unresolved health problem. Rev Inst Med Trop Sao Paulo. 2012; 54 (2):69–75. PMID: 22499419

12. Meny P, Menendez C, Quintero J, Hernandez E, Rios C, Balassiano IT, et al. Characterization of *Lep-tospira* isolates from humans and the environment in Uruguay. Rev Inst Med Trop Sao Paulo. 2017; 59: e79. https://doi.org/10.1590/S1678-9946201759079 PMID: 29267587

13. Meny P, Menendez C, Ashfield N, Rios C, Iglesias T, Quintero J, et al. Leptospirosis in human groups at risk in Uruguay. In: Society International Leptospirosis, editor. 10th International Leptospirosis Society Conference 2017 "Science for People"; Palmerston North, New Zealand 2017. p. 181.

14. Libonati H, Pinto PS, Lilenbaum W. Seronegativity of bovines face to their own recovered leptospiral isolates. Microb Pathog. 2017; 108:101–103. https://doi.org/10.1016/j.micpath.2017.05.001 PMID: 28478204

15. Chideroli RT, Pereira UP, Goncalves DD, Nakamura AY, Alfieri AA, Alfieri AF, et al. Isolation and molecular characterization of *Leptospira borgpetersenii* serovar Hardjo strain Hardjobovis in the urine of naturally infected cattle in Brazil. Genet Mol Res. 2016; 15(1): gmr8473.
16. Cosate MRV, Sakamoto T, de Oliveira Mendes TA, Moreira EC, Regis da Silva CG, Brasil B, et al. Molecular typing of *Leptospira interrogans* serovar Hardjo isolates from leptospirosis outbreaks in Brazilian livestock. BMC Vet Res. 2017; 13(1):177. https://doi.org/10.1186/s12917-017-1081-9 PMID: 28619055

17. Salgado M, Otto B, Moroni M, Sandoval E, Reinhardt G, Boqvist S, et al. Isolation of *Leptospira interrogans* serovar Hardjoprajitno from a calf with clinical leptospirosis in Chile. BMC Vet Res. 2015; 11:66. https://doi.org/10.1186/s12917-015-0369-x PMID: 25888965

18. Myers DM, Jelambi F. Isolation and identification of *Leptospira* Hardjo from cattle in Argentina. Trop Geogr Med. 1975; 27(1):63–70. PMID: 49113

19. Director A, Penna B, Hamond C, Loureiro AP, Martins G, Medeiros MA, et al. Isolation of *Leptospira interrogans* Hardjoprajitno from vaginal fluid of a clinically healthy ewe suggests potential for venereal transmission. J Med Microbiol. 2014; 63(Pt 9):1234–1236. https://doi.org/10.1099/jmm.0.065466-0 PMID: 24934563

20. Carmona-Gasca CA, León Lara L, Castillo-Sánchez LO, Ramírez-Ortega JM, Ko A, Luna Palomera C, et al. Detection of *Leptospira santarosai* and *L. kirschneri* in cattle: new isolates with potential impact in bovine production and public health. Vet Mex. 2011; 42(4):277–288.

21. Faine S, Adler B, Bolin C, Perolat P. *Leptospira* and leptospirosis. 2nd ed. Melbourne: MediSci; 1999. 272 p.

22. Fouts DE, Matthias MA, Adhikarla H, Adler B, Amorim-Santos L, Berg DE, et al. What makes a bacterial species pathogenic?: comparative genomic analysis of the genus *Leptospira*. PLoS Negl Trop Dis. 2016; 10(2):e0004403. https://doi.org/10.1371/journal.pntd.0004403 PMID: 26890609

23. Galloway RL, Hoffmaster AR. Optimization of LipL32 PCR assay for increased sensitivity in diagnosing leptospirosis. Diagn Microbiol Infect Dis. 2015; 82(3):199–200. https://doi.org/10.1016/j.diagmicrobio. 2015.03.024 PMID: 25912810

24. Hamond C, Martins G, Loureiro AP, Pestana C, Lawson-Ferreira R, Medeiros MA, et al. Urinary PCR as an increasingly useful tool for an accurate diagnosis of leptospirosis in livestock. Vet Res Commun. 2014; 38(1):81–85. https://doi.org/10.1007/s11259-013-9582-x PMID: 24222053

25. Ahmed N, Devi SM, Valverde Mde L, Vijayachari P, Machang'u RS, Ellis WA, et al. Multilocus sequence typing method for identification and genotypic classification of pathogenic *Leptospira* species. Ann Clin Microbiol Antimicrob. 2006; 5:28. https://doi.org/10.1186/1476-0711-5-28 PMID: 17121682

26. Merien F, Amouriaux P, Perolat P, Baranton G, Saint Girons I. Polymerase chain reaction for detection of *Leptospira* spp. in clinical samples. J Clin Microbiol. 1992; 30(9):2219–2224. PMID: 1400983

27. Salaun L, Merien F, Gurianova S, Baranton G, Picardeau M. Application of multilocus variable-number tandem-repeat analysis for molecular typing of the agent of leptospirosis. J Clin Microbiol. 2006; 44 (11):3954–3962. https://doi.org/10.1128/JCM.00336-06 PMID: 17088367

28. Ahmed A, Thaipadungpanit J, Boonsilp S, Wuthiekanun V, Nalam K, Spratt BG, et al. Comparison of two multilocus sequence based genotyping schemes for *Leptospira* species. PLoS Negl Trop Dis. 2011; 5(11):e1374. https://doi.org/10.1371/journal.pntd.0001374 PMID: 22087342

29. Jolley KA, Maiden MC. BIGSdb: Scalable analysis of bacterial genome variation at the population level. BMC Bioinformatics. 2010; 11:595. https://doi.org/10.1186/1471-2105-11-595 PMID: 21143983

30. Chideroli RT, Goncalves DD, Suphoronski SA, Alfieri AF, Alfieri AA, de Oliveira AG, et al. Culture strategies for isolation of fastidious *Leptospira* serovar Hardjo and molecular differentiation of genotypes Hardjobovis and Hardjoprajitno. Front Microbiol. 2017; 8:2155. https://doi.org/10.3389/fmicb.2017. 02155 PMID: 29163438

31. Hamond C, Pestana CP, Medeiros MA, Lilenbaum W. Genotyping of *Leptospira* directly in urine samples of cattle demonstrates a diversity of species and strains in Brazil. Epidemiol Infect. 2016; 144 (1):72–75. https://doi.org/10.1017/S0950268815001363 PMID: 26076668

32. Martins G, Loureiro AP, Hamond C, Pinna MH, Bremont S, Bourhy P, et al. First isolation of *Leptospira noguchii* serogroups Panama and Autumnalis from cattle. Epidemiol Infect. 2015; 143(7):1538–1541. https://doi.org/10.1017/S0950268814002416 PMID: 25185756

33. Silva ÉF, Cerqueira GM, Seyffert N, Seixas FK, Hartwig DD, Athanazio DA, et al. *Leptospira noguchii* and human and animal leptospirosis, Southern Brazil. Emerg Infect Dis. 2009; 15(4):621–623. https:// doi.org/10.3201/eid1504.071669 PMID: 19331754

34. Adler B, Moctezuma AD. *Leptospira* and leptospirosis. Vet Microbiol. 2010; 140(3-4):287–296. https:// doi.org/10.1016/j.vetmic.2009.03.012 PMID: 19345023

35. Miraglia F, de Morais ZM, Dellagostin OA, Seixas FK, Freitas JC, Zacarias FG, et al. Molecular and serological characterization of *Leptospira interrogans* serovar Canicola isolated from dogs, swine, and bovine in Brazil. Trop Anim Health Prod. 2013; 45(1):117–121. https://doi.org/10.1007/s11250-012- 0181-6 PMID: 22610538
36. Levett PN. Leptospirosis. Clin Microbiol Rev. 2001; 14(2):296-326. https://doi.org/10.1128/CMR.14.2.296-326.2001 PMID: 11292640

37. Arent ZJ, Gilmore C, San-Miguel Ayanz JM, Neyra LQ, Garcia-Pena FJ. Molecular epidemiology of *Leptospira* serogroup Pomona infections among wild and domestic animals in Spain. Ecohealth. 2017; 14(1):48-57. https://doi.org/10.1007/s10393-017-1210-8 PMID: 28213654

38. Hathaway SC, Marshall RB, Little TW, Headlam SA, Winter PJ. Differentiation of reference strains of leptospires of the Pomona serogroup by cross-agglutination absorption and restriction endonuclease analysis. Res Vet Sci. 1985; 39(2):145-150. PMID: 2999927

39. Bolin CA, Zuerner RL. Correlation between DNA restriction fragment length polymorphisms in *Leptospira interrogans* serovar Pomona type Kennewicki and host animal source. J Clin Microbiol. 1996; 34(2):424-425. PMID: 8789028

40. Ellis WA. Leptospirosis. In: Zimmerman J. J., Ramirez A., Schwartz K. J., Stevenson G. W., editors. Diseases of Swine. 10th ed. Hoboken,NJ: Wiley-Blackwell; 2012. p. 770-778.

41. Ellis WA. The diagnosis of leptospirosis in farm animals. In: Ellis W.A., Little T.W.A., editors. The Present State of Leptospirosis Diagnosis and Control. Dordrecht, The Netherlands: Martinus Nijhoff Publishers; 1986. p. 13-24.

42. Silva EF, Santos CS, Athanazio DA, Seyffert N, Seixas FK, Cerqueira GM, et al. Characterization of virulence of *Leptospira* isolates in a hamster model. Vaccine. 2008; 26(31):3892-3896. https://doi.org/10.1016/j.vaccine.2008.04.085 PMID: 18547690

43. Pinto PS, Loureiro AP, Penna B, Lilenbaum W. Usage of *Leptospira* spp. local strains as antigens increases the sensitivity of the serodiagnosis of bovine leptospirosis. Acta Trop. 2015; 149:163-167. https://doi.org/10.1016/j.actatropica.2015.05.008 PMID: 25997883

44. Vallee E, Ridler AL, Heuer C, Collins-Emerson JM, Benschop J, Wilson PR. Effectiveness of a commercial leptospiral vaccine on urinary shedding in naturally exposed sheep in New Zealand. Vaccine. 2017; 35(9):1362-1368. https://doi.org/10.1016/j.vaccine.2016.04.037 PMID: 27109564

45. Bolin CA, Alt DP. Use of a monovalent leptospiral vaccine to prevent renal colonization and urinary shedding in cattle exposed to *Leptospira borgpetersenii* serovar Hardjo. Am J Vet Res. 2001; 62(7):995-1000. PMID: 11453500

46. Zuerner RL, Alt DP, Palmer MV, Thacker TC, Olsen SC. A *Leptospira borgpetersenii* serovar Hardjo vaccine induces a Th1 response, activates NK cells, and reduces renal colonization. Clin Vaccine Immunol. 2011; 18(4):684-691. https://doi.org/10.1128/CVI.00288-10 PMID: 21288995

47. Thornley CN, Baker MG, Weinstein P, Maas EW. Changing epidemiology of human leptospirosis in New Zealand. Epidemiol Infect. 2002; 128(1):29-36. PMID: 11895088

48. Naiman BM, Alt D, Bolin CA, Zuerner R, Baldwin CL. Protective killed *Leptospira borgpetersenii* vaccine induces potent Th1 immunity comprising responses by CD4 and gammadelta T lymphocytes. Infect Immun. 2001; 69(12):7550-7558. https://doi.org/10.1128/IAI.69.12.7550-7558.2001 PMID: 11705932

49. Naiman BM, Blumerman S, Alt D, Bolin CA, Brown R, Zuerner R, et al. Evaluation of type 1 immune response in naive and vaccinated animals following challenge with *Leptospira borgpetersenii* serovar Hardjo: involvement of WC1(+) gammadelta and CD4 T cells. Infect Immun. 2002; 70(11):6147-6157. https://doi.org/10.1128/IAI.70.11.6147-6157.2002 PMID: 12379692

50. Adler B. Vaccines against leptospirosis. Curr Top Microbiol Immunol. 2015; 387:251-272. https://doi.org/10.1007/978-3-662-45059-8_10 PMID: 25388138