Use of the Coding Region of Leptospira Sp. LigB C-terminus as a Marker for Diagnostics of Animal Leptospirosis

The causal agent of leptospirosis, pathogenic strains of the genus Leptospira spp., has Outer Membrane Proteins (OMPs) on its surface which play a fundamental role in infection and pathogenesis. Studies on the genome showed that the LigB protein gene is conserved in different pathogenic species. Methods The aim of this work was to propose a new end point PCR based on the amplication of the LigB C-Terminal coding region (ligb-ct), never used before and conserved among pathogenic Leptospira spp. Eighteen reference pathogenic, 2 intermediate and 2 no-pathogenic strains of Leptospira spp. were used. DNA from 10 other microorganism species were included in this study to determine the analytical specicity. We obtained 100% positivity for pathogenic Leptospira strains. We found no cross-reaction with intermediate and non-pathogenic strains or with other microorganisms, highlighting a high analytical specicity. Analytical sensitivity estimated on clinical samples was higher on serum than blood and urine (6-9 x 10 2 lept/ml and 6-9 x 10 5 and 6-9 x 10 6 leptospires/ml, respectively). Multiple sequence alignment of this region in positive Leptospira species conrmed a high degree of conservation, with only a few single nucleotide polymorphisms.


Results
We obtained 100% positivity for pathogenic Leptospira strains. We found no cross-reaction with intermediate and non-pathogenic strains or with other microorganisms, highlighting a high analytical speci city. Analytical sensitivity estimated on clinical samples was higher on serum than blood and urine (6-9 x 10 2 lept/ml and 6-9 x 10 5 and 6-9 x 10 6 leptospires/ml, respectively). Multiple sequence alignment of this region in positive Leptospira species con rmed a high degree of conservation, with only a few single nucleotide polymorphisms.

Conclusion
To the best of our knowledge, the LigB C-Terminal coding region has not been previously used for molecular diagnostic and could be used for early diagnosis of leptospirosis.

Background
Leptospirosis is an antropozoonotic endemic disease worldwide, mainly in underveloped countries with high levels of poverty. It is caused by pathogenic strains of Leptospira spp., characterized by fever and multi-organ failure in humans and animals. In addition, reproductive problems such as abortion and infertility in production animals as a consequence of these infections, result in important economic losse [1,2].
The taxonomy of the genus was rst based on serology, which distinguished almost 300 serovars.
According to a more recent characterization using genetic molecular taxonomy, at least 64 species divided into 2 phylogenetic clades (P and S) and 4 subclades (P1, P2, S1 and S2) were described [3].
The pathogen has a variety of Outer Membrane Proteins (OMPs), such as LigA and LigB lipoproteins, on its surface, which mediate interaction with host extracellular matrix proteins, allowing the bacteria to colonize multiple host organs [4,5].
LigA and LigB belong to the superfamily of bacterial immunoglobulin-like (Big) repeat domain proteins, shared with adhesins of other bacteria, such as intimin and invasin from enteropathogenic E. coli and Yersinia spp., respectively [6,7].
Exposure to physiological osmolarity induces leptospires to express high levels of the Lig surface proteins and increases adhesion between leptospires and the host extracellular matrix and plasma proteins, such as collagens, laminin, bronectin and brinogen [8]. Unlike LigA, LigB contains 12 Big domains followed by a long carboxy-terminal region, exposed to the extracellular medium and its are expressed early in the course of infection [9].
Lig genes are highly conserved (70-99% identity) among pathogenic species and virulent pathogenic leptospiral isolates [10,11], however, the ligB gene is present in all pathogenic leptospira, while the ligA gene is only found in L. interrogans and L. kirschneri strains, and both are absent in intermediate or saprophyte Leptospira spp. species [12,13].
Despite its relevance, leptospirosis is one of the most under-diagnosed zoonotic diseases. The Micro Agglutination Test (MAT) is considered the gold standard and is the most used tool for serological diagnosis. However, it presents certain inconveniences such as the requirement of paired samples with intervals of 15-21 days to have a con rmatory result, and lack of detection in the acute phase of the disease due to the absence of detectable antibodies in blood [1].
As a complement to MAT, PCR has been lately used for the molecular detection of pathogenic Leptospira DNA using different target genes such as secY and ab [14], ompL1 [15], lipL32 [16,17], and ligB [|18,19]. However, few of the described PCR tests were subjected to rigorous validation analysis in the human and veterinary areas.
The aim of this work was to propose a new end point PCR based on the ampli cation of the LigB C-Terminal coding region (ligb-ct) as a novel diagnostic tool for leptospirosis.

Samples
Twenty-two reference Leptospira spp. strains were used in this study, 18 of which were pathogenic, 2 nonpathogenic and 2 intermediate (Table 1). Four strains were isolated from clinical samples from dogs (serovar Canicola strain Hond Utrecht IV), bovines (serovar strain Pomona), and 2 from rodents (serovar Copenhageni strain M20 and serovar Fiocruz strain L1-130), from Argentina. Environmental samples were isolated from water from the Argentine localities of Puerto Iguazú, Misiones province (1 sample), and Añatuya, Santiago del Estero province (3 samples). Water was pre-ltered through Whatman lter paper before ltration through 0.22 milipore size membranes. An aliquot (1 ml) of each ltered sample was inoculated in Fletcher medium supplemented with 5-Fluorouracil and Neomycin.  [20], following the modi cations of Johnson and Harris [21] and bacterial growth was monitored under a dark-eld microscope.
Genomic DNA extraction was performed using 20 µl sample and 150 µl Chelex-100 (Bio-rad USA) as described Hamer et al. [22], and incubated at 56 °C during 20 min followed by 8 min at 100 °C. Samples were centrifuged at 10,000 rpm for 5 s and 20 µl of the supernatant were collected and stored at -20 °C until use.
The integrity of the extracted DNA was analyzed by electrophoresis in 1% agarose gels. DNA quantities were estimated by absorbance measurements in a Nanodrop spectrophotometer.
The PCR ampli cation reaction was performed in a nal volume of 50 µl using 5 µl puri ed DNA template (approximately 250 ng). The PCR mixture contained 1X buffer (500 mM KCl/100 mM Tris/HCl, pH 9.0), 3 mM MgCl 2 , 0.5 µmol for each primer, 0. The analytical sensitivity was determined using serially diluted from L. interrogans serovar Pomona, in samples of bovine serum, blood and urine. The initial leptospiral concentration was adjusted by spectrophotometric measurements at 420 nm to approximately 6-9 × 10 8 bacteria/ml (corresponding to OD 420 = 0.365).

Sequencing and alignment
After veri cation of the presence of amplicons by electrophoresis, 9 PCR products were puri ed and sequenced by Macrogen (Korea). The Clustal Omega program [23] was used to carry out a multiple

Results
The end point LigBct PCR was performed under the conditions mentioned in Materials and Methods. Ampli cation of a 700-800 bp fragment, compatible with the expected 726 bp fragment, was observed in all of the pathogenic Leptospira spp. species used in this study, while no ampli cation was obtained in intermediate and non-pathogenic leptospires (Fig. 2). Also, a single band was observed for all clinical and environment DNA samples used in this study. DNA integrity of negative samples was controlled by agarose gel electrophoresis and LipL32 gene PCR ( Analytical sensitivity analysis showed signi cant differences in a controlled assay in which bovine serum, blood, and urine were experimentally infected with serial dilutions of a L. interrogans serovar Pomona suspension. After DNA extraction and LigBct PCR, detection limits for serum, blood and urine were 6-9 × 10 2 , 6-9 × 10 5 and 6-9 × 10 6 leptospires/ml, respectively.

Discussion
Leptospirosis remains a signi cant public health problem in many countries. Initially, the diagnosis of leptospirosis was based on the isolation of leptospires from clinical samples or the demonstration of seroconversion in serum samples [24]. Although these tools are still used, molecular methods based on DNA ampli cation such as PCR, also play an important role for diagnosis. PCR often proves to be superior to traditional methods for the detection of carrier and sick animals, due to its speed and high sensitivity and speci city [25,26].
The rst end point PCR to amplify Leptospira sp. DNA was developed by Gravekamp in 1993 using the secY and aB genes of pathogenic leptospires as targets and was applied to blood samples [14]. Although this PCR was reported not to ampli y the secY sequence of non-pathogenic species, Palaniappan ampli ed DNA from the non-pathogen L. bi exa, which could lead to false positive results in clinical samples [19].
The LipL32 protein gene is also widely used as target for diagnosis of leptospirosis in clinical samples. A Taqman qPCR developed by Stoddard [16] based on this gene can detect 10 1 leptospires/ml blood and 10 3 leptospires/ml serum, while a qPCR developed by Levett 17 has an analytical sensitivity of 3 genome equivalents per reaction and approximately 10 genome equivalents in human urine.
Studies of the genome of the Leptospira genus showed that the lipL32 gene is present not only in all species belonging to the pathogenic group, but also in recently described low-virulence species such as the intermediate group [27,28,12].
The ligB gene has also been used as PCR target. In 2017, Benacer et al [29] developed a duplex PCR targeting the 16S ribosomal RNA subunit gene (rrs) and different regions of the ligB gene. This assay detects pathogenic species DNA with a sensitivity of 10 3 leptospires/ml of urine or water. Ali et al. [30] also worked with the ligB gene and developed a Loop Mediated Isothermal Ampli cation (LAMP) protocol, using six pairs of primers targetting a segment from nt 72 to nt 290. This LigB-LAMP assay was applied to urine samples from cattle and dogs and proved to be highly sensitive, detecting a minimum of 150 fg DNA. However, the cost of reagents for LAMP is higher than that of PCR, making LAMP more di cult to implement in some diagnostic laboratories. In 2018, Martinez et al. [18] proposed a typing tool based on the ampli cation of a 1044 bp fragment of the ligB gene, from 1158 nt to 2203 nt, which could discriminate some serovars.
In the present work, the LigB-C terminal coding region (ligb-ct) is used for the rst time as PCR target, and proves useful for the diagnosis of animal leptospirosis. No cross-reaction with other microorganisms commonly found in clinical laboratory samples was observed, and no ampli cation was obtained with intermediate Leptospira species. This indicates that this tool is highly speci c for pathogenic Leptospira species.
The sensitivity of the LigBct PCR was higher for serum samples than for blood and urine samples. This could be because anticoagulants used in blood extraction can act as PCR inhibitors affecting the e ciency of PCR when a commercial extraction kit is not used [31].
Importantly, since serum samples are used in the MAT gold standard, LigBct PCR could be a complementary tool to detect traces of leptospiral DNA in this type of samples when there are positive cases with low antibody titers. In addition, the successful ampli cation of various species of pathogenic leptospires achieved, as shown in Table 1, indicates that the primers recognize conserved regions and can be used for diagnosis.
Multiple sequence alignment of the LigB C-terminus coding region of 9 strains of pathogenic leptospires showed the presence of 17 single nucleotide polymorphisms (SNPs) ( Table 2). Since different nucleotides were observed at some of these polymorphic sites when two or more samples of the same serovar were analyzed, these SNPs cannot be used as a genotyping tool. The observed polymorphism within one serovar in the 3' LigB-C terminal coding region may be due to the fact that the encoded protein segment is extracellular and exposed to the selection pressure of the host immune system, thus this region is not a marker of neutral selection.
We showed that the ligBct PCR could successfully amplify DNA of environmental samples, after isolating and cultivating leptospires under laboratory conditions. Future studies will be devoted to test whether this assay can be applied to amplify Leptospira spp. DNA from raw water samples without cross-reaction with other microorganisms present in the sample and not tested in the present work.

Conclusion
The analytical speci city and analytical sensitivity of a diagnostic PCR is given by the success of ampli cation on the pool of samples used. The results of this PCR targeting the 3' region of the LigB-C terminal coding region are encouraging for the detection of leptospiral DNA in clinical samples in the initial phase of leptospirosis. LigBct PCR could be applied as a screening tool when a leptospire infection is suspected, or as a prophylaxis tool for early control in areas where there is a history of leptospirosis clinical cases. This PCR could also be a useful complement to MAT, the reference method worldwide, as it showed high sensitivity in bovine serum samples. Availability of data and materials All relevant data are within the paper and its Supporting Information les