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

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 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.


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 as epidemic outbreaks, causing severe, sometimes fatal disease in both humans and animals [7,8].
Since the first systematic studies in [1960][1961][1962][1963][1964][1965][1966][1967][1968][1969][1970], serologic studies in animals have repeatedly shown high prevalence of exposure to Leptospira in Uruguay, with individual seropositivity in the 25-50% range, and herd prevalence figures of 50-70% [9,10]. Leptospirosis is considered as a re-emerging bovine disease in Uruguay since 1998 [10], after what stricter epidemiologic surveillance policies have been adopted by governmental agencies. Human leptospirosis has been included into the official list of diseases of mandatory notification. Leptospirosis in Uruguay is endemic, with limited epidemic outbreaks in rural areas. The annual incidence of human leptospirosis is estimated at 15 per 100,000 [11], with precise figures not determined due to under-reporting and extremely scarce systematic studies in southern Latin America of morbidity/mortality burden [7]. The human disease appears to be associated with bovine infection, as well as to rainfalls and floods [11], with recent isolation efforts revealing the presence of three L. interrogans serovars, two L. kirschneri and one L. borgpetersenii [12,13].
Despite the relevance of bovine leptospirosis as a cause of bovine abortions and infertility in Uruguay, there have been no extensive studies on the actual identities of Leptospira species and serovars obtained from animals in the field. There are currently no repositories of autochthonous isolates available in the public domain, thus constraining vaccine companies to the use of foreign strains as vaccine antigens. Even though Hardjo serovars have been suspected for years to be involved in bovine infection cases [2,14], to the best of our knowledge only four L. interrogans and two L. borgpetersenii isolates belonging to this serovar have been reported in South America [15][16][17] obtained in Brazil and Chile. An early study also reported six Hardjo isolates in Argentina, without distinguishing the species [18], and two isolates of L. interrogans Hardjo were also reported, one in sheep from Brazil [19] and one in cattle from Mexico [20]. We now report the first results of a multicentric effort, over the course of 3 years, aimed at isolating pathogenic Leptospira strains in Uruguay, from infected cattle in the field and at abattoirs. A detailed serologic and genetic characterization of such isolates uncovers a larger than expected variety of Leptospira species and serovars. These data will be instrumental for the design of better bacterin vaccines, as well as for improving diagnosis and epidemiologic studies in Uruguay and neighboring South American countries.

Ethics statement
Urine and blood sampling from cattle in the field were performed by professional veterinarians, respecting international recommendations for animal welfare, with approval granted by the Ethics Committee for the Use of Animals for Experimentation (Comisión de Etica en el Uso de Animales de Experimentación CEUA), DILAVE, Ministry of Livestock, Agriculture and Fishery (Ministerio de Ganaderia, Agricultura y Pesca MGAP), Uruguay, according to national law #18,611. Permission to take samples for the study was received from the animal owners and the abattoirs.

Identification of herds suspected of leptospirosis, and field urine and blood sampling
Forty-eight herds from both dairy and beef farms were sampled in this study, during a 33-month period (Jan 2015-Sep 2017). Private veterinarians who suspected the disease sent the first samples to our laboratory at the Ministry of Livestock, Agriculture and Fishery. Following current protocols in Uruguay, serum samples from 12 animals from each suspected herd, were 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 Leptospira 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 cutoff 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 Isolation of Leptospira strains from Uruguayan cattle 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 !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][23][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 PCRamplification 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 MgCl 2 , 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´-TTCCCCCCATTGAGCAAGATT-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. interrogans, and L. borgpetersenii species, as previously described [27].

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 prioritize cultures (Fig 1), identifying those samples that proved positive for pathogenic Leptospira spp. A strong inhibitory effect on lipL32 PCR amplification was frequently observed, dependent 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. santarosai), 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 confirmed 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 pathogenic 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 threshold. Following up with this approach at the herd level, 77% of the farms (37/48) that were studied, harbored !1 animal(s) excreting pathogenic Leptospira.

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 overgrowth 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).
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 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).

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 genomospecies identified by partial rrs gene sequencing: L. interrogans, L. borgpetersenii, L. kirschneri 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. interrogans 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 homogeneous 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 Uruguay, as well as a number of other strains obtained from both human and animal origin in several 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 Kennewicki (serogroup Pomona), our most frequent bovine isolate, has actually been also recovered 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 disseminate the infection to exposed humans.
The successful culture of leptospires from bovine samples has likely been boosted by optimizing field sampling protocols, especially after quantifying time-dependent Leptospira viability 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.
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 Leptospira 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 identified as L. noguchii, but more information is urgently needed in order to establish the contribution 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. interrogans 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 interesting to note that the isolates belonging to L. interrogans and L. borgpetersenii, displayed limited 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 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: Ã serovar Pomona ÃÃ serovar Panama ÃÃÃ serovar Autumnalis ÃÃÃÃ 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 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, Tindependent 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. 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. 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-Neil 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)