Produção Nacional
Revisado por pares
An overview on the molecular diagnosis of animal leptospirosis
2020; Oxford University Press; Volume: 72; Issue: 5 Linguagem: Inglês
10.1111/lam.13442
ISSN1472-765X
AutoresMaria Isabel Nogueira Di Azevedo, Walter Lilenbaum,
Tópico(s)Leptospirosis research and findings
ResumoLetters in Applied MicrobiologyVolume 72, Issue 5 p. 496-508 Review ArticleFree Access An overview on the molecular diagnosis of animal leptospirosis M.I.N. Di Azevedo, Laboratory of Veterinary Bacteriology, Biomedical Institute, Fluminense Federal University, Niterói, Rio de Janeiro, BrazilSearch for more papers by this authorW. Lilenbaum, Corresponding Author wlilenbaum@id.uff.br orcid.org/0000-0002-2434-8728 Laboratory of Veterinary Bacteriology, Biomedical Institute, Fluminense Federal University, Niterói, Rio de Janeiro, Brazil Correspondence Walter Lilenbaum, Laboratory of Veterinary Bacteriology, Biomedical Institute, Fluminense Federal University, Niterói, Rio de Janeiro, Brazil. E-mail: wlilenbaum@id.uff.brSearch for more papers by this author M.I.N. Di Azevedo, Laboratory of Veterinary Bacteriology, Biomedical Institute, Fluminense Federal University, Niterói, Rio de Janeiro, BrazilSearch for more papers by this authorW. Lilenbaum, Corresponding Author wlilenbaum@id.uff.br orcid.org/0000-0002-2434-8728 Laboratory of Veterinary Bacteriology, Biomedical Institute, Fluminense Federal University, Niterói, Rio de Janeiro, Brazil Correspondence Walter Lilenbaum, Laboratory of Veterinary Bacteriology, Biomedical Institute, Fluminense Federal University, Niterói, Rio de Janeiro, Brazil. E-mail: wlilenbaum@id.uff.brSearch for more papers by this author First published: 17 December 2020 https://doi.org/10.1111/lam.13442Citations: 1AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinked InRedditWechat Abstract The most common presentation of animal leptospirosis is the subclinical and silent chronic form, that can lead to important reproductive disorders. The diagnosis of this chronic form remains a challenge. The aim of the present study is to gather and critically analyse the current information about molecular tools applied to animal leptospirosis diagnosis, particularly the silent chronic presentation of the infection. Regarding clinical specimens, samples from urinary tract were the most used (69/102, 67·7%), while few studies (12/102, 11·8%) investigated samples from reproductive tract. Concerning the molecular methods applied, the most used is still the conventional polymerase chain reaction (PCR) (46/102, 45%), followed by real-time PCR (38/102, 37·2%). The lipL32 gene is currently the most common target used for Leptospira detection, with 48% of studies applying this genetic marker. From all the studies, only few (21/102, 20·5%) performed gene sequencing. According to the majority of authors, current evidence suggests that lipL32-PCR is useful for an initial screening for Leptospira DNA detection in animal clinical samples. Posteriorly, if DNA sequencing could be performed on positive lipL32-PCR samples, we encourage the use of secY gene as a genetic marker. The molecular methods appear as the most important tools for the diagnosis of the chronic silent leptospirosis on domestic animals, reinforcing its evident impact not only on animal reproduction but also on a One Health context. Introduction The genus Leptospira belongs to the phylum Spirochaetes, order Spirochaetales, family Leptospiraceae (Adler 2015). They are slow-growing and aerobic bacteria (Li et al. 2000), with unique morphological characteristics, such as helix shape and well-developed endoflagel, providing high mobility to the spirochetes (Picardeau 2013). Two classification schemes are used for leptospires. The first is based on serology and is useful for epidemiological purposes, defining serogroups and serovars. The other uses molecular taxonomy for species identification, also known as genomospecies (Guernier et al. 2018). Currently, based on phylogenetic analysis, Leptospira is divided in three lineages that reflects the level of pathogenicity: saprophytic (n = 26), intermediate (n = 21) and pathogenic (n = 17) (Vincent et al. 2019). It is important to highlight that serological and genetic classifications are not associated, and different serovars may belong to the same genomospecies (Levett 2015). Since it affects many domestic animals as well as wildlife and also humans, leptospirosis is classified as a zoonosis (Lim 2011). Animal infection most frequently occurs through direct contact with Leptospira contaminated urine or indirectly from interaction with contaminated water and/or soil (Guernier et al. 2018). As the bacterium has been detected in reproductive fluids of bovines (Masri et al. 1997; Loureiro et al. 2017), equines (Hamond et al. 2014; Hamond et al. 2015), ovine (Lilenbaum et al. 2008; Director et al. 2014; Nogueira et al. 2020) and swine (Fernandes et al. 2020), sexual transmission should not be neglected. Additionally, transmission also can occur through reproduction biotechnologies, like embryo transfer and artificial insemination (Bielanski and Surujballi 1998; Givens 2018), if antibiotics are not used in the process. Human leptospirosis is primarily characterized by acute clinical features known as Weil’s disease, with intense signs as fever, icterus, renal insufficiency and mortality (O'Toole et al. 2015). Domestic animals may also present this acute, potentially lethal infection, similar to Weil’s disease. In those cases, significant renal damage can occur, particularly in dogs. Horses present recurrent uveitis (Verma et al. 2012) and decreased athletic performance (Hamond et al. 2012). In bovine, as well as in other ruminants, the acute and severe form of leptospirosis is uncommon and frequently associated with sporadic outbreaks in calves caused by incidental strains (Loureiro and Lilenbaum 2020). Indeed, the most common presentation of animal leptospirosis is the subclinical and silent chronic form, which is very often neglected (Adler 2015). In this presentation, reproductive signs are predominant. As a consequence of Leptospira colonization of the reproductive tract, this chronic form determines important reproductive disorders, leading to impacting economic losses (Mori et al. 2017). Loureiro and Lilenbaum (2020) recently suggested that the silent reproductive form of leptospirosis should be considered as a specific syndrome, so called bovine genital leptospirosis. It is most often caused by adapted strains from the Sejroe serogroup, and is associated to early embryonic losses and consequent oestrus repetition, very probably due to uterine inflammation and/or direct invasion of the embryo by leptospires (Mori et al. 2017; Libonati et al. 2018). More recently, it was demonstrated a high oxidative damage in sheep infected with leptospires, contributing to reproductive disturbance pathophysiology (Silva et al. 2019). Similar to other ruminants, abortion is the most important clinical consequence of leptospiral infection in caprine (Dehkordi and Taghizadeh 2012). In horses, reproductive disorders include abortion and stillbirth, while the birth of infected foals is a common sequel (Whitwell et al. 2009). Moreover pregnancy losses have been attributed to a local inflammation caused by the presence of leptospires in the uterine environment (Pinna et al. 2013). Leptospirosis is widely reported in dogs and only recently chronic infection has been studied. It also includes reproductive failure (Johnston and Raksil 1987; Rossetti et al. 2005; Reagan and Sykes 2019), but in a less prevalent way than that seen in ruminants. Sant'Anna et al. (2019) demonstrated that chronic kidney disease may be associated with asymptomatic leptospiral infection in dogs in endemic regions. Feline stillbirths associated to Leptospira infection have also been described (Reilly et al. 1994). The correct diagnosis of animal leptospirosis depends on some important factors, such as collection of adequate specimens and mainly the stage of infection. Currently, diagnosis may be accomplished by detection of the bacterium through direct observation of leptospires, culture or its DNA detection; and indirectly by detection of specific antibodies (serology) (Adler 2015). It is important to emphasize that, while the first test group reflects infection, the second predicts previous exposition. Direct visualization of leptospires in blood, urine and milk of infected animals can be performed using darkfield microscopy, but sensitivity and specificity are low, so this method is not routinely used in the diagnosis of animal leptospirosis (Ellis 2015; Reagan and Sykes 2019). A positive culture of biological samples (blood, urine, tissue) is the definitive proof of infection, but culturing leptospires is laborious and fastidious. The bacterium requires special growth media and incubation can last for months. Although highly specific, sensitivity of culturing is low, what may be related to low bacteraemia, administration of antibiotics prior to specimen collection, or technical difficulties (Reagan and Sykes 2019). Regarding indirect diagnosis, serology, mainly the microscopic agglutination test (MAT), is adequate for diagnosing acute leptospirosis in humans and dogs, where infected patients present high IgM and IgG titers that are easily detected, and its use is indicated by OIE for diagnostic purposes (OIE 2012). Its epidemiological value resides in its ability to predict the circulating serogroups. However, this assay requires significant expertise to perform and interlaboratory variation in results is high, so results must be interpreted with caution (Loureiro and Lilenbaum 2020). Moreover even though useful for a collective herd diagnosis, serology present important limitations for chronic animal leptospirosis, and is not adequate for an individual diagnostic. In a study conducted with samples of different hosts, it was shown that serological and molecular results are very often discrepant, and many infected animals may present seronegative. In this context, polymerase chain reaction (PCR) should be considered and encouraged as a useful tool for an accurate diagnosis of leptospirosis in livestock (Hamond et al. 2014a,b). Molecular diagnostic tools for leptospiral DNA detection, such as PCR, has been widely applied for animal leptospirosis diagnosis, with the main advantage of presenting high sensitivity and specificity (Hamond et al. 2014). The test does not require the presence of viable organisms and an early diagnosis can be performed, since bacterial DNA can be detected even before the development of a serologic response to infection (Waggoner and Pinsky 2016). Several genetic markers have been applied for pathogenic Leptospira detection and identification, with different levels of taxonomic resolution. Latest, real-time PCR (qPCR) has been applied, what provides a faster and more sensitive diagnosis compared to traditional PCR (Picardeau 2013). One main limitation of molecular diagnosis in animal leptospirosis is the absence of standardization, since the few studies that carried out genetic characterization applied different genetic markers, and comparison of results becomes a challenge. Although the diagnosis of the acute disease is reasonably standardized both for the direct and indirect methods, the diagnosis of the silent chronic form of animal leptospirosis remains a challenge. The mild, often inapparent symptomatology makes a clinical suspicious very rare. In most domestic animals infected, Leptospira shedding is intermittent and not very intense; besides, they present low antibody titres (Nally et al. 2018), making difficult both direct as indirect diagnosis. In addition, despite reproductive problems are widely reported, due to the classic association with disorders in urinary tract and the typical Weil disease in humans, the majority of studies focus on bacterial investigation on kidneys/urine. In contrast, genital samples remain underused, when, in fact, should be the primary specimen of choice for the diagnosis of chronic form of animal leptospirosis, mainly on ruminants (Loureiro and Lilenbaum 2020). Based on that context, the aim of the present study is to gather and critically analyse current information about molecular tools applied to animal leptospirosis diagnosis, particularly the silent chronic presentation of the infection. Results and Discussion Clinical specimens Identification of pathogenic Leptospira species by PCR is more efficient when performed on bacterial isolates obtained from infected animals (once DNA is more concentrated, intact and free of contaminants), but, as previously exposed, culturing leptospires is laborious. The identification and, mainly, typing of leptospires directly from clinical samples without culturing is a challenge. The standard typing method, multi locus sequence typing, that uses sequence polymorphisms of multiple housekeeping genes for characterization and investigation of evolutionary relationships among closely related bacteria, in the vast majority of times can only be performed with total efficiency when based on isolates, after culturing (Varni et al. 2014). Thus, efforts have been made to perform genetic investigations directly from clinical samples using short DNA regions (Guernier et al. 2018). Examples of successful Leptospira sp. DNA detection from noncultured clinical samples are shown in Table S1 and summarized on Fig. 1a. Figure 1Open in figure viewerPowerPoint Distribution of studies involving animal leptospirosis according to clinical specimen (a) and molecular methodology applied (b). Scrawled areas correspond to studies that performed DNA sequencing. [Colour figure can be viewed at wileyonlinelibrary.com] Regarding the best specimens for analysis, it is consensus that acute/septicaemic disease should include blood and urine testing (Reagan and Sykes 2019). As well, for the diagnosis of the Weil’s disease, urine is universally accepted as the best specimen for PCR (Haake and Levett 2015). Occasionally, organs are tested (Fang et al. 2015; Jawor et al. 2017; Grippi et al. 2020), but it cannot be performed on large scale on animals and it usually requires post-mortem samples for diagnosis. Nevertheless, for the molecular diagnosis of the reproductive syndrome, it was previously shown that the status of genital leptospiral carriers cannot be reliably predicted by the detection of the agent in urine (Loureiro et al. 2017; Silva et al. 2019). As example, in an outbreak in which 24 dairy cows suffered reproductive problems due to leptospirosis, only one was PCR positive using urine as specimen, while eight cows had leptospiral DNA detected in vaginal discharge samples (Pimenta et al. 2019). So, the use of specimens from reproductive tract, such as cervico-vaginal mucus and uterine fragments for Leptospira DNA investigations should be encouraged (Di Azevedo et al. 2020). Recently, Loureiro and Lilenbaum (2020) proposed a method for collection of vaginal discharge from livestock that avoid contamination with urine. It consists in the collection of cervico-vaginal mucus from the anterior region of the urinary meatus, specifically the vaginal fornix, using a sterile cytology brush. Despite that, analysing the studies that performed molecular diagnosis of animal leptospirosis, the great majority of them used samples from the urinary tract (kidney and urine) (69/102, 68%), while only 12/102 (11%) investigated the reproductive tract (semen, vaginal fluid, uterine fragment). Aborted tissues, blood and other specimens (e.g. liver, ocular fluids and respiratory tissues) were analysed in 12/102 (12%), 3/102 (3%) and 6/102 (6%) of the studies respectively (Table S1, Fig. 1a). Methods Regarding the molecular methods applied for animal leptospirosis diagnosis, the most used is still the conventional PCR (46/102, 45%), followed by qPCR (38/102, 37%). Despite its greater sensitivity, the real time PCR is more expensive, and require an equipment that is not always available. Traditional PCR is cheaper, so more applied. Loop-mediated isothermal amplification (LAMP), Nested-PCR and others (FRET-PCR, HRMA, LSSP-PCR, PCR-RFLP) have also been applied, but discreetly, each corresponding to 8/102 (7·8%) of studies (Table S1, Fig. 1b). Conventional PCR (PCR) has been applied for animal leptospirosis diagnosis since the 90's and then it has been extensively applied worldwide (Table S1). Reactions targeting lipL32 are the most commonly conducted, on 28/45 studies (62·2%). It presents the advantage of detecting leptospiral DNA in a great variety of clinical specimens (Table S1) and in a more specific basis. The relatively rapid and standardized protocol for lipL32 amplification justifies its wide use, at least as a screening tool for diagnosis purposes. Real-time PCR (qPCR) is faster than conventional PCR and less sensitive to contamination (Picardeau 2013). The assays have improved sensitivity and are less prone to contamination compared with conventional PCR. It has been widely applied for animal leptospirosis diagnosis studies (37/102, 36·2%), including bovine (Alinaitwe et al. 2019), cats (Weis et al. 2017; Sprißler et al. 2019; Alashraf et al. 2020), dogs (Rojas et al. 2010; Delaude et al. 2017; Miotto et al. 2018; Spangler et al. 2020; Le Guyader et al. 2020), horses (Pearce et al. 2007; Hamond et al. 2015; Erol et al. 2015; Sauvage et al. 2019), sheep (Fang et al. 2015; Vallée et al. 2015; Benkirane et al. 2016) and swine (Verma et al. 2015). A high sensitivity has been achieved using diverse clinical specimens (urine, blood, ocular tissues, vaginal fluid, kidney) and different molecular targets (lipL32, rrs, secY, gyrB) (Table S1). As an alternative DNA amplification method, in the last few years the so-called loop-mediated isothermal amplification (LAMP) have been applied (7/102, 6·8%) (Table S1, Fig. 1b). It presents high specificity, since it uses Bst DNA polymerase which has strand displacement DNA synthesis activity along with inner, outer and loop specific primers that create cauliflower-like structures with multiple loops (Ali et al. 2017). Unlike thermocycler-based PCR methods, the LAMP reaction can be performed in a constant temperature water bath, and the amplification of a target DNA sequence is achieved in approximately one hour (Suwancharoen et al. 2016). Moreover the reaction can be observed by naked eyes with visual fluorescence. This simplicity and low cost enhance its applicability for on-field screening of pathogens and for limited resourced laboratories (Monica et al. 2019). Regarding its use as a diagnostic tool for animal leptospirosis, it has been successfully applied for detecting leptospiral DNA on cattle and buffalo (Suwancharoen et al. 2016; Chadsuthi et al. 2018), dogs (Gentilini et al. 2017) and other domestic animals (Tubalinal et al. 2018). Molecular targets for diagnostic tests PCR-based tests have focused on both universally gene present in bacteria, as gryB, rrs (16S rRNA gene) and secY; and surface proteins restricted to Leptospira, such as lipL21, lipL32, lipL41 and ligB (Guernier et al. 2018). The 16S rRNA gene was the first genetic marker applied for Leptospira identification and has been used since a long time as a target for many diagnostic PCR assays (Smythe et al. 2002), and a representative number of studies have applied it so far (25/102, 24·5%) (Table S1, Fig. 2a). It has a good capacity to discriminate between pathogenic, intermediates and saprophytic leptospires, but presents low taxonomic resolution to differentiate between Leptospira species within a clade, being useful only for an initial screening. Despite that, some recent studies still use it as genetic target (Table S1, Fig. 2b). Nevertheless, it is important to highlight that, although extremely useful in the first studies on PCR for animal leptospirosis and its huge importance and pioneering, currently other targets with a better discriminatory power should be prioritized. Figure 2Open in figure viewerPowerPoint Distribution of studies involving animal leptospirosis according to genetic marker applied (a) and its use along the years (b). [Colour figure can be viewed at wileyonlinelibrary.com] In contrast, a short region of the lipL32 gene is currently the most common target used for Leptospira detection, with 49/102 (48%) of studies applying this genetic marker (Table S1, Fig. 2a). The lipL32 gene encodes an outer membrane lipoprotein that originally was first referred to be present in the pathogenic species but absent in the nonpathogenic (Stoddard et al. 2009). Nowadays, despite its large use, it is known that it can also be found on intermediate strains (Picardeau 2017). Regarding sensitivity, when compared to rrs gene, it was shown that lipL32 is a suitable target for hybridization probe-based qPCR assays (Gentilini et al. 2015). The secY gene is a housekeeping gene located on the CI chromosome that encodes a pre-protein translocase important for the export of proteins across the cytoplasmic membrane (Durack et al. 2015; Haake and Levett 2015), and, among the less conserved genes, has been the most used (11/102, 10·7%) (Fig. 2a), from 2010 onwards (Fig. 2b). Although some studies used that gene for the primary diagnosis of animal Leptospira infections (Benkirane et al. 2016; Denipitiya et al. 2017), it may lead to false-positive results since it is also present in other bacteria, like Staphylococcus sp. or coliforms. It presents a good discriminatory power, and sequence analysis of the gene allows identification of species, strains and occasionally, genotypes. In this context, it is currently more used at a second step for discriminatory and taxonomic purposes, providing important epidemiological information. In a recent meta-analysis performed to evaluate the diagnostic accuracy of genetic markers for the detection of Leptospira in human and animal clinical samples, secY gene exhibited higher diagnostic accuracy measures compared with other genetic markers (Lam et al. 2020). Through secY gene sequencing, Hamond et al. (2015) assessed genotype diversity of Leptospira species from urine of cattle. Later, Pires et al. (2018) identified L. interrogans on paraffined bovine uteri, suggesting its possible association with the physiopathogenesis of reproductive failure. Similarly, L. interrogans was identified as the most common agent of the genital infection in mares with reproductive problems (Hamond et al. 2015). Importantly, regarding investigation of chronic infection, L. interrogans was identified and associated to renal histological alterations in asymptomatic sheep in a Brazilian slaughterhouse (Almeida et al. 2019). More recently, secY sequencing allowed the identification of four different species circulating in cattle of the Brazilian Amazon (Guedes et al. 2019). This same gene was used for characterizing as L. interrogans Sejroe serogroup amplicons from uteri of cows (Di Azevedo et al. 2020). One bacterial gene that is also used in phylogenetic studies is the gyrB gene, which is reported to have a higher nucleotide divergence in Leptospira species than the 16S RNA rrs gene (Huang 1996; Slack et al. 2006). Even discreetly, this gene target has also been used for animal leptospirosis primary diagnosis (5/102, 4·9%) (Table S1, Fig. 2a). An epidemiological investigation in a dairy farming in New Zealand was performed in heifers through gyrB-qPCR diagnosis (Yupiana et al. 2019). In the same region, using the same methodology, shedding of pathogenic Leptospira sp. in sheep and cattle was assessed (Fang et al. 2015) and a high prevalence of infected animals was detected on sheep (Vallée et al. 2015). Although few studies have applied the flaB gene for animal leptospirosis diagnosis (4/102, 3·9%) (Table S1, Fig. 2a), this target has been used to discriminate between Leptospira species. It encodes a flagellum protein, and has been successfully used to identify other bacterial species including Campylobacter (Harrington et al. 2003) and Borrelia (Lin et al. 2004). Restriction fragment length polymorphism of the PCR product of this gene was successfully used to discriminate between Leptospira species in the laboratory (Kawabata et al. 2006) and was also used in field settings to study the molecular epidemiology of canine leptospirosis in Japan (Koizumi et al. 2013). Gamage et al. (2014) applied flaB -PCR to assess the carrier status of leptospirosis among cattle in Sri Lanka and found three pathogenic species, L. borgpetersenii (50%), L. kirschneri (35%) and L. interrogans (15%). Similarly, sequence analysis of Leptospira flaB in large ruminants revealed the formation of three major clusters with L. borgpetersenii or L. kirschneri and molecular epidemiology based on this gene clarified the main source of leptospires for other farms (Villanueva et al. 2016). The RNA polymerase β-subunit gene rpoB is used in phylogenetic analyses of several bacterial genera, and was proposed for Leptospira typing to circumvent the limitations of the 16S rRNA discrimination (La Scola et al. 2006). Bioinformatics studies later confirmed its high value for discrimination (Cerqueira et al. 2010), and its utility in epidemiological studies was demonstrated by studies in Brazil and India using Leptospira isolates from human and animals (Jorge et al. 2012; Balamurugan et al. 2013). For animal leptospirosis diagnosis purposes, its use is still restrained, and only three studies (2·9%) obtained a successful identification direct from clinical samples (Table S1, Fig. 2a), all of them performed after 2018 (Fig. 2b). Zaidi et al. (2018) suggested that dogs are maintenance hosts for zoonotic leptospirosis after genetic characterization based on rpoB sequences analysis. More recently, for diagnostic purposes, leptospiral rpoB gene sequencing from canine urine samples revealed L. interrogans, indicating implications on animal and public health (Spangler et al. 2020). Noteworthy to highlight that, from all the studies aiming animal leptospirosis diagnosis, only very few (21/102, 20·5%) performed gene sequencing (Please see the scrawled areas on graph bars of Fig. 2b). This is particularly a problem when genes common to various bacteria are applied for primary diagnosis (e.g. secY, gyrB, flab and rpoB). Moreover we strongly encourage gene sequencing, not only for the correct species definition, but also for DNA database (GenBank and others) enrichment. The results from the present revision clearly show that, from the less conserved genes not exclusive to Leptospira, secY has been the most successfully amplified and sequenced directly from clinical specimens, but only after primary diagnosis using lipL32 as target (Table S1, Fig. 2a). Other genes, like gyrB, flab and rpoB, despite its good taxonomic application based on isolates, have not yet been proven effective in clinical specimens to date, and further studies are necessary to validate its applicability. Molecular diagnosis of animal chronic disease As seen before, lipL32-PCR has been widely applied for primary detection of leptospiral DNA on urinary and/or reproductive tract of domestic animals and detected chronic infected animals in an individual level. Using lipL32 as PCR target, Loureiro et al. (2017) found a high frequency (50·4%) of leptospiral vaginal carriers among slaughtered cows, highlighting the role of vaginal carriers and indicating that venereal transmission (female-to-male) could occur. It has been also been successfully used on paraffin-embedded tissue samples (Pires et al. 2018) and in abattoirs, revealing risk of exposure among workers (Alinaitwe et al. 2019). Regarding dogs, a high number of asymptomatic dogs were positive in an endemic area (Sant'anna et al. 2017), and chronic infection was associated with canine chronic kidney disease (Sant'Anna et al. 2019). Other studies have provided identification of pathogenic Leptospira on urine (Rojas et al. 2010; Delaude et al. 2017; Miotto et al. 2018; Altheimer et al. 2020; Spangler et al. 2020; Le Guyader et al. 2020) and formalin-fixed paraffin-embedded liver (McCallum et al. 2019). Most of recent studies conducted on cats also use this genetic marker for diagnosis purposes, performing qPCR from urine samples (Weis et al. 2017; Sprißler et al. 2019; Alashraf et al. 2020). lipL32-PCR was also applied to horses and clinical samples include urine (Hamond et al. 2012b; Hamond et al. 2013; Hamond et al. 2014; Hamond et al. 2015; Pinna et al. 2014), vaginal fluid (Hamond et al. 2014; Hamond et al. 2015) and ocular fluid (Sauvage et al. 2019). Potential for venereal transmission has been proposed on sheep after lipL32-PCR detection of pathogenic Leptospira on vaginal fluid of a clinically healthy ewe (Director et al. 2014). A high frequency of genital carriers of Leptospira sp. were detected in sheep slaughtered in a semi-arid region of northeastern Brazil (Silva et al., 2019) and the importance of genital transmission route was later reinforced (Nogueira et al. 2020). Similarly, chronic infection by Leptospira sp. in asymptomatic sheep was demonstrated by lipL32-PCR (Almeida et al. 2019). In summary, according to the majority of authors, current evidence suggests that lipL32-PCR is useful for an initial screening for Leptospira DNA detection in animal clinical samples. Posteriorly, if DNA sequencing could be performed on positive lipL32-PCR samples, we encourage the use of secY gene as genetic marker. Beyond its good taxonomic resolution, providing satisfactory diagnostic accuracy measures, database regarding this gene is relatively extensive (more than 1500 sequences available), including different species and serogroups, wh
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