Human deaths due to infectious diseases remain the leading scourge of humanity [1]. Many of these emerging infectious diseases are zoonotic, originating from both domestic and wild animals [2]. Leptospirosis, a leading yet neglected zoonotic bacterial disease, is responsible for widespread morbidity and disproportionately impacts people in regions with warm, humid climates in which the bacteria can easily proliferate [3]. Difficulties in diagnosing human and animal leptospirosis remain an important problem, and despite the availability of molecular diagnostics, diagnosis still largely depends on archaic serology [4]. The disease is caused by Gram-negative spirochetes of the genus Leptospira, which has 64 genomic species and 300 serovars identified to date [5]. A wide variety of animals—rodents, livestock, and wild animals—are sources of leptospiral infection by excreting Leptospira in their urine. Humans are accidental hosts and acquire the infection from either direct or indirect sources such as contaminated water, like rivers, puddles, and rice paddies [6,7,8,9,10,11]. Animal leptospirosis is of public health and economic importance as it negatively impairs livestock production and represents an infectious threat to humans; dogs, which both serve as companion animals and roam ferally, are susceptible to leptospiral infection in diverse settings. Cattle leptospirosis can reflect in abortion, stillbirth, premature birth, general reproductive failure, and milk production drop syndrome [12]. Bovines are susceptible to the infection from multiple serovars and Leptospira species, including L. borgpetersenii serovar Hardjo and L. interrogans serovar Pomona [13,14]. Infected animals may be seronegative and still excrete Leptospira [13]. The serious health effect on livestock is an important motivation to develop a pan-leptospiral vaccine that would be long-lasting and provide sterile immunity and cross-protection among diverse species and serovars [15]. To achieve a successful vaccination process, it is very important to understand the risk and assessments of the disease in a population.

A human being who comes into contact with urine-infected animals or contaminated environments, whether directly or indirectly, is considered at risk for leptospiral infection [19]. Finding a way to prevent and treat this disease is always one of the goals of researchers working with these bacteria. To date, naturally acquired anti-leptospiral immunity has been considered primarily to be antibody-mediated, with immune responses directed against leptospiral lipopolysaccharide (LPS) [20,21,22]. However, it also appears that cell-mediated responses contribute to protection against some serovars such as Hardjo in cattle. In animals, such as dogs, there is currently no gold standard methodology for in vitro prediction of experimental in vivo evaluation of vaccine efficacy [23]. In the case of recombinant vaccines, several vaccine approaches have been tested, including bacterial DNA, viral delivery vaccines, live attenuated bacteria, and subunit vaccines [24]. In the complex extensive reverse vaccinology study to date, a total of 238 where proteins identified and evaluated as potential vaccine candidates [25]. A hamster colonization model was used to evaluate pool of recombinant proteins (5 proteins/pools) and >70% were immunogenic [25]. However, none of the recombinant protein pools conferred protection against kidney colonization [25]. In metanalysis research involving vaccines and vaccination for dogs, researchers estimated a general 84% protection against carrier status [23]. The American Veterinary Medical Association explains that currently available vaccines effectively prevent leptospirosis and protect dogs for at least 12 months [26,27,28]. Annual vaccination is recommended for at-risk dogs and required for bovines. Leptospirosis is a nearly silent infection, rarely inducing outward clinical signs in cattle, as was reflected in peripheral blood mononuclear cell profiles [29].

With few outwardly observable clinical signs, cattle are a chronic host of serologically identical but genetically distinct members of serovar Hardjo, namely L. borgpetersenii serovar Hardjo (type Hardjo-bovis) and L. interrogans serovar Hardjo (type Hardjo-prajitno) [12]. Cattle are the major reservoir of these agents, which can infect humans and other animals and cause acute disease [30,31]. In North America, L. borgpetersenii serovar Hardjo type Hardjo-bovis is most often isolated from cattle [13,31]. Vaccination with bacterins does reduce livestock-human transmission, and it can also reduce the impact of leptospirosis on cattle, which improves animal production [29]. Information is a key feature in the prevention of diseases like leptospirosis. Recently, in research with VM proteins, Chaurasia et al. [32] found that vaccination of C3H/HeJ mice with L. interrogans serovar Lai VM proteins protected mice from any clinical manifestations of the disease. The authors observed that this led to ~3–4 log¹⁰ reduction in bacterial load in the liver and kidney, two key organs in the pathogenesis of leptospirosis and transmission of Leptospira, respectively. Hence, recombinant protein vaccines may be key to global immunization against pathogenic leptospirosis, eliminating kidney colonization and spread in the environment [32].

Virulence in Leptospira still has several unknown aspects. Fouts et al. [16] described virulence in terms of survival mechanisms of Leptospira bacteria such as adhesion to the extracellular matrix (ECM), complement evasion and ECM degradation via metalloproteases, motility, chemotaxis, resistance to oxidative stress. As well evading/motility mechanism like the immunodominant proteins of Leptospira and the exotoxin PF07598 paralogous gene family. Members of the PF07598 gene family are expressed and upregulated to various extents, as shown in vivo in a hamster model of acute leptospirosis. It has been established that group 1 (pathogenic Leptospira) includes L. interrogans serovars Icterohaemorrhagiae and Copenhageni, which are the strains most often associated with severe and fatal outcomes [5]. The members of this group uniquely encode in their genomes virulence-related protein families, such as the metalloproteases-associated protein family and the VM protein family [16], which suggests the potential importance of these protein families in the pathogenesis of leptospirosis [5]. An ultimate objective for clinical leptospirosis research is to combat the lack of wide cross-protection provided by current serogroup/serovar-specific bacterin vaccines by developing a pan-leptospirosis vaccine [33,34,35]. The premise of much ongoing research in the field is that identifying pathogenic virulence factors conserved across species/serovar/strain will enable the use of such proteins for vaccine development to target host immune responses that protect against numerous serovars of Leptospira [33].

Virulence factors are often the focus of characterization since a bacterin vaccine that exposes an immunized host to more known virulence factors are likely to produce improved protection [33,34,35]. To date, only a limited number of known virulence factors have been identified in pathogenic Leptospira, and they have been more intensely studied in L. interrogans than in L. borgpetersenii [16,33]. The outer membrane, surface-exposed virulence factors are of particular focus as they interact directly with the host during infection and are thus available to antibodies [33].

The large novel gene family PF07598, previously of unknown function, encodes the VM proteins found uniquely in group 1 pathogenic Leptospira [17,18,36]. The identification of these new virulence-associated genes should spur additional experimental inquiry into their potential roles in mediating Leptospira pathogenesis and, perhaps, in enabling Leptospira to persist in the hostile and competitive environmental microbial world [17].

Two of the largest phylogenetically distinct pathogenic species are Leptospira borgpetersenii and Leptospira interrogans, which, combined, cause most cases of leptospirosis [37]. Although the clinical symptoms of infection due to these two species are similar, they are transmitted differently; epidemiological data support a host-to-host transmission cycle for L. borgpetersenii [38]. After the ongoing ecological niche switch from free-living to a symbiotic lifestyle (concomitant with gene expansion), this group of bacteria stabilizes and restricts their lifestyle in specific niches [39]. Overall, so far, is not clear if is the reduction in the number of virulence-modifying proteins in L. borgpetersenii associated with a switch to a symbiotic lifestyle that causes protein production changes, although they remain as virulent as L. interrogans, which has more VM proteins [12,13,14,15].

In a study comparing L. borgpetersenii and L. interrogans survival in water at 20 °C, the former lost >90% viability within 48 h whereas L. Interrogans retained 100% viability over the same period [38]. L. interrogans retained 30% viability over a 3-week incubation, by which time no viable L. borgpetersenii were detected (R.L.Z. unpublished data) [38]. The authors concluded that L. borgpetersenii does not tolerate nutrient deprivation and does not survive passage through water [38]. L. borgpetersenii has a limited capacity to acquire nutrients and survive in environments external to a mammalian host [38]. Although the reason for this is not yet completely clear, it is possible that the large range of potential hosts that can be infected by Leptospira species requires some specificity and that horizontal gene transfers may be one of the methods allowing fast adaptation to these hosts [39]. Adherence is only one of the possible mechanisms underlying the host specificity [40]. In some instances, the ability of a pathogen to infect a host has been correlated with its ability to adhere to cells from that species [40]. This leads to more evolutionary questions about the Leptospira species and host dynamics.

The body of work has shown that the PF07598 gene family is restricted to group 1 pathogenic Leptospira and is not found in either intermediate or saprophytic Leptospira [16,17,18,36,41]. VM proteins encode the paralogs, which vary in their numbers in L. interrogans serovar Lai (12), L. interrogans serovar Copenhageni (13), L. borgpetersenii serovar Hardjo (4), and several others using system biology and “pathogenomic” approaches [16]. This raises several questions. For instance, why does L. borgpetersenii have four VM proteins? There may be one protein and three isomers, or there may be four different proteins. Perhaps there have also been mildly deleterious mutations or, similarly, new insertions of intrinsic elements that might facilitate intragenomic recombination.

A tenet of evolution is that alleles that are under negative selection are often deleterious and confer no evolutionary advantage; these are removed from the gene and are eventually extinguished from the population [42]. Conversely, alleles under positive selection do confer an evolutionary advantage and lead to an increase in the overall fitness of the organism; thus, they increase in frequency until they eventually become fixed in the population [42]. Comparing the complete genome of two strains of L. borgpetersenii serovar Hardjo, one hypothesis is that the bacteria are undergoing a process of insertion sequence (IS)-mediated genome reduction [38], leading simultaneously to the perception of genome reduction in L. borgpetersenii and genome expansion in L. interrogans, which reflects differences in the environments during transmission between hosts. That is one theory that could support the evolution of L. borgpetersenii. Based on several comparisons [16,17,18,43], the interpretation is that Lai has orthologs in L. interrogans Copenhageni (LIC) apart from LIC_10639, and each protein from L. borgpetersenii has copies inside the species. Answering the general question about why this happens is a work in progress.

6. Antigenicity and Epitopes