Summary

Leptospirosis is an important and often ignored disease affecting more than one million people a year worldwide, from which 50,000 die. No serovar is known to be adapted to humans, so humans acquire the disease directly from animals (contact with urine or tissues) or indirectly (contact with urine contaminated water). The three main environments showing transmission risks are water-based, rodent-borne and livestock/pet-borne environments. Leptospirosis occurs worldwide but it is more frequently found in humid tropical climates, especially in regions with poor sanitary infrastructure, deficient drainage, and as a result large numbers of rats. The contact with natural bodies of water (wet farming) increases the risk of infection. Outbreaks tend to occur during rainy seasons and it has been associated with flooding. The disease has a broad geographical distribution due to the large spectrum of hosts including domestic and wild mammals. Leptospirosis affects risk groups that are exposed to animal reservoirs or urine contaminated environments and people in contact with natural bodies of water. Leptospirosis is regarded as the most widespread zoonosis.

Leptospirosis is caused by spirochetes of the genus Leptospira which comprises 20 species that are phylogenetically separated into 3 clusters: 9 pathogenic, 6 saprophytic, and 5 intermediate. The etiologic agents of this disease are pathogenic and intermediate species of Leptospira which comprise more than 260 serovars. Pathogenic and intermediate clusters have been reported to cause infection and in this text we will refer to them as parasitic Leptospira. The organism is a thin spirochete of 0.1 to 0.2 µm in diameter and 6 to 12 µm in length, so that it can be filtered through 0.45 µm filters. It is very motile showing translational motility, travelling approximately 20 μm in 2 to 3 seconds in ordinary media.

Leptospirosis is considered a neglected disease, and the spirochete causing this disease, Leptospira is difficult to recover in culture and only recently molecular genetics tools have been developed suitable for the study of this spirochete. These factors have contributed to a lag in the understanding of its virulence mechanisms, its immunogenic properties and finally a lag in the development of vaccines that may induce strong and durable protection.

To date, different methods for inactivation of leptospires for use as vaccines have been applied, these include formalin, phenol, ethanol, heat, freeze and thaw and radiation; and after 100 years the only licensed vaccines are whole leptospira inactivated bacterins. These have been used to immunize animals, mainly dogs, cattle and pigs. because of problems with components of the culture media and reactogenicity, such bacterins have not been widely accepted for use in humans, however human bacterin vaccines have been used successfully in several regions, including China, Japan, Cuba, and Europe. No live vaccines are currently licensed.

Studies of parasitic Leptospira in the environment are very limited mainly because it is difficult to isolate these spirochetes from surface waters or soil without getting contamination with saprophytic Leptospira. However, genomic studies suggest that parasitic Leptospira evolved from an environmental (free living) Leptospira found in water or mud, similarly to the contemporary members of the saprophytic cluster (such as L. biflexa). There seems to be some evolutionary variation within parasitic leptospires; some species (such as L. interrogans) have retained genes associated to environmental survival whereas other parasitic leptospires (such as L. borgpetersenii) seem to have lost these genes. Leptospiral survival in water is bolstered by viscous material such as agar (and possibly biofilm). Some strains of parasitic Leptospira are able to produce biofilms when incubated in fresh water even under the absence of nutrients, furthermore L. interrogans (Pathogenic cluster) has been found in nature associated to biofilms formed by environmental bacteria. Some parasitic leptospira are able to produce biofilm when incubated in distilled water (low nutrient conditions) and seemed to detect nutrients such as fatty acids causing the dispersion (release) of leptospiral cells from the biofilm. It has been observed that leptospiral biofilm has a five to sixfold increase in antibiotic resistance in all the strains used. It is tempting to speculate that biofilms may protect pathogenic Leptospira against other toxic compounds in the environment. The role of biofilm in the transmission of Leptospira to susceptible hosts are still unclear.

Soil may also play a role in environmental survival; some researchers have shown that Leptospira survived in the wet soil on dry days and appeared in the surface water on rainy days, suggesting that soil could be a reservoir for leptospires in the environment. Even though leptospires are considered as fragile spirochetes, studies have shown that they can survive and even maintain their virulence despite unfavorable conditions such as cold and nutrient-poor acidic waters after up to 20 months.

There is no standard protocols to culture parasitic species of Leptospira from bodies of fresh water or soil. There are only few reports describing isolation of parasitic Leptospira from environmental samples and the reports show little success; the main difficulty in the isolation of these species is the overgrowth of environmental bacteria or the fast-growing saprophytic Leptospira. Molecular techniques are a lot more promising at detecting parasitic Leptospira in the environment. Many PCR protocols have been developed using mainly genes that are present only in pathogenic Leptospira such as the hemolysis-associated protein-1gene, lipL32, or pathogenic specific sequences of genes such as flaB, secY, and 16S ribosomal gene. The main limitation of many of these analyses is that they fail to detect parasitic Leptospira of the intermediate cluster which are commonly found in water. Detection of genomic DNA sequences do not guaranty the viability of bacteria in water or other samples. RNA methods are under development to detect viable bacteria.

Pathogen(s)

Leptospirosis is a deadly bacterial infection caused by pathogenic members of the genus Leptospira. Human leptospirosis has long been recognized as an environmental disease, with documented reports dating at least a century ago [START_REF] Noguchi | The Survival of Leptospira (Spirochaeta) Icterohaemorrhagiae in Nature; Observations Concerning Microchemical Reactions and Intermediary Hosts[END_REF]. Today, it is clearly acknowledged that most human infections originate from environmental exposure to water and soils contaminated by the urine excreted by infected animals. Human exposure to contaminated water mostly occurs through bathing or swimming in natural freshwater bodies, but isolated contamination of water supplies has also been involved in outbreaks of leptospirosis (reviewed in [START_REF] Levett | Leptospirosis[END_REF].

Leptospirosis is very challenging to diagnose clinically. Educating clinicians about considering leptospirosis when patients present with acute febrile syndromes, particularly when there has been recent exposure to freshwater, could greatly improve diagnosis. Although leptospires are considered to be very fragile bacteria, being highly susceptible to a number of physical and chemical agents [START_REF] Faine | Leptospira and Leptospirosis[END_REF], they present an astonishing capacity to survive in freshwater ecosystems for weeks to months. However, little is known about the fate of pathogenic leptospires in water, despite the importance of such knowledge towards preventing this potentially fatal bacterial infection in humans.

There are three main reasons for limited knowledge about the fate of leptospires in water. First, the genus Leptospira comprises both virulent species, which are the etiological agents of human and animal leptospirosis, and saprophytic species, which are non-virulent normal inhabitants of freshwater ecosystems. Second, the taxonomy of the genus has long been based on serology, which distinguished almost 300 serovars grouped into more than 23 serogroups, but only two species (Leptospira interrogans sensu lato, which grouped all virulent species, and Leptospira biflexa sensu lato for all saprophytic species until the 1980s). The two previous species grouping of the 1980s have since been declared invalid, based on more recent characterization using genetic molecular taxonomy, which to date has discriminated at least 35 species [START_REF] Brenner | Further determination of DNA relatedness between serogroups and serovars in the family Leptospiraceae with a proposal for Leptospira alexanderi sp. nov. and four new Leptospira genomospecies[END_REF][START_REF] Ramadass | Genetic characterization of pathogenic Leptospira species by DNA hybridization[END_REF][START_REF] Yasuda | Deoxyribonucleic acid relatedness between serogroups and serovars in the family Leptospiraceae with proposals for seven new Leptospira species[END_REF]Thibeaux et al., 2018b;Thibeaux et al., 2018a). In addition, another group of Leptospira (named "intermediates") has also been described [START_REF] Schmid | Newly recognized Leptospira species ("Leptospira inadai" serovar lyme) isolated from human skin[END_REF][START_REF] Perolat | Leptospira fainei sp. nov., isolated from pigs in Australia[END_REF], which currently incorporates 11 species with at least some displaying some degree of virulence. Early scientific reports did not always describe whether the leptospires studied had some virulence potential. The older literature therefore needs to be interpreted with caution to consider these taxonomical differences. Third, and finally, yet as importantly, pathogenic strains of Leptospira are slowgrowing, and can easily become overgrown by saprophytic species and other microorganisms, even with selective culture medium. Still, the body of literature published over the last century is proving useful in building some knowledge about the occurrence, persistence and survival of leptospires in water. Putting this knowledge together can help improve prevention of this significant, possibly reemerging, environmental disease.

Global Burden of Disease

Global distribution

One of the first descriptions of the disease now known as leptospirosis was reported by Larrey in 1812, during a blockade of Napoleon's troops in Cairo; and more than a century ago Adolph Weil described a severe form of the disease, since known as Weil's disease. Leptospirosis is an important and often ignored disease affecting more than one million people per year worldwide, with 58,900 deaths [START_REF] Costa | Global Morbidity and Mortality of Leptospirosis: A Systematic Review[END_REF][START_REF] Ko | Leptospira: the dawn of the molecular genetics era for an emerging zoonotic pathogen[END_REF]. Leptospirosis occurs worldwide, but it is more frequently found in humid tropical climates, especially in regions with poor sanitary infrastructure, deficient drainage, and as a result, large numbers of a key animal reservoir: rats. While inappropriate management of human excreta may support rodent densities and thereby be a contributing factor, human infections are often associated with exposures to natural bodies of water (wet farming). Outbreaks tend to occur during rainy seasons and it has been associated with flooding (Della Rossa et al., 2016;[START_REF] Ganoza | Determining risk for severe leptospirosis by molecular analysis of environmental surface waters for pathogenic Leptospira[END_REF][START_REF] Trevejo | Epidemic leptospirosis associated with pulmonary hemorrhage-Nicaragua[END_REF][START_REF] Concepcion-Acevedo | Initial Public Health Laboratory Response After Hurricane Maria -Puerto Rico, 2017[END_REF]Reardon, 2017). The disease has a broad geographical distribution due to the large spectrum of hosts, including domestic and wild mammals, including rats and mice, which harbor the bacterium in their kidneys and release it through their urine (Carmona Gasca et al., 2013;[START_REF] Dietrich | Leptospira and Bats: Story of an emerging friendship[END_REF][START_REF] Ko | Leptospira: the dawn of the molecular genetics era for an emerging zoonotic pathogen[END_REF]. Various animal hosts are further discussed in Section 1.3.2. Leptospirosis affects risk groups that are exposed to animal reservoirs or urinecontaminated environments, such as veterinarians, abattoir workers, sewage workers, military personnel, people in contact with natural bodies of water, and increasingly including individuals partaking in water sports and recreation [START_REF] Agampodi | Outbreak of leptospirosis after white-water rafting: sign of a shift from rural to recreational leptospirosis in Sri Lanka[END_REF][START_REF] Katz | Leptospirosis in Hawaii, 1974-1998: epidemiologic analysis of 353 laboratory-confirmed cases[END_REF][START_REF] Ko | Leptospira: the dawn of the molecular genetics era for an emerging zoonotic pathogen[END_REF][START_REF] Lau | Leptospirosis: an emerging disease in travellers[END_REF][START_REF] Morgan | Outbreak of leptospirosis among triathlon participants and community residents in Springfield, Illinois[END_REF][START_REF] Sejvar | Leptospirosis in "Eco-Challenge" athletes, Malaysian Borneo[END_REF]. Leptospirosis is regarded as the most widespread zoonotic disease [START_REF] Evangelista | Leptospira as an emerging pathogen: a review of its biology, pathogenesis and host immune responses[END_REF] and is reemerging in regions once thought to be relatively free of the pathogen, such as northern Sweden [START_REF] Strand | Highly Pathogenic Leptospira Found in Urban Brown Rats (Rattus norvegicus) in the Largest Cities of Sweden. Vector Borne Zoonotic DisVector Borne Zoonotic DisVector[END_REF] (see Table 1 below). 1.1.2 Symptomatology (morbidity and case-fatality ratios)

The symptoms of leptospirosis vary from asymptomatic to a severe hemorrhagic and/or multi-organ disease. The severity of the disease depends on the Leptospira species [START_REF] Alston | Leptospiral Jaundice Among Sewer-workers[END_REF] and the ability of the strain to cause infection. The disease can be divided in two stages; in the first stage (septicemic phase), leptospires multiply in the host's blood and other tissues and cause fever, chills, headache, anorexia, myalgia (mainly in calves) and nausea or fulminant cases, and in the immune phase conjunctival suffusion and jaundice. Headache, photophobia, nausea, retro-orbital pain may indicate the presence of Leptospira in the cerebrospinal fluid [START_REF] Picardeau | Diagnosis and epidemiology of leptospirosis[END_REF]. Severe leptospirosis (Weil's disease or severe pulmonary hemorrhage syndrome) can develop after the septicemic phase. Weil's disease is characterized by hemorrhage, 1.0 Epidemiology of the Disease and jaundice, and renal failure [START_REF] Bharti | Leptospirosis: a zoonotic disease of global importance[END_REF], while severe pulmonary hemorrhage is linked to massive intraalveolar bleeding. Mortality due to these 2 syndromes may range from 5-15% and renal failure may affect 16-40% of cases. Pulmonary pathology may occur in 20-70% of the cases and symptoms may range from cough, dyspnea, hemoptysis, to respiratory distress syndrome [START_REF] Adler | Leptospira and leptospirosis[END_REF]. Death can occur from kidney failure, pulmonary hemorrhage, or other serious multiorgan system dysfunction.

Taxonomic Classification of the Agent(s)

Leptospirosis is caused by spirochetes of the genus Leptospira, which currently comprises 35 species that are phylogenetically separated into three clusters: 13 pathogenic, 11 saprophytic, and 11 intermediate [START_REF] Adler | Leptospira and leptospirosis[END_REF]Thibeaux et al., 2018b;Thibeaux et al., 2018a). The etiologic agents of this disease are pathogenic and intermediate species of Leptospira, which comprise more than 260 serovars (Adler, 2015a). Pathogenic and intermediate clusters have been reported to cause infection and, in this Chapter, we will refer to them as "virulent" Leptospira. The organism is a thin spirochete of 0.1 to 0.2 µm in diameter and 6 to 12 µm in length, so it may even pass through 0.22 µm filters. Hence 0.22 µm prefiltration is often used to decontaminate Leptospira cultures from other bacteria [START_REF] Evangelista | Leptospira as an emerging pathogen: a review of its biology, pathogenesis and host immune responses[END_REF]. It is a highly motile bacterium showing translational motility, travelling approximately 20 μm in 2 to 3 seconds in ordinary media. It has been demonstrated that such a motility is due to its coiled periplasmic flagellum that rotates at the leading end, generating a helical wave that propels the cell by rolling in an direction opposite to that of the flagellar gyration [START_REF] Goldstein | Motility of the spirochete Leptospira[END_REF]Charon, 1988, Picardeau et al., 2001).

Transmission

Routes of transmission

Leptospira, as a fragile spirochete, is not resistant to desiccation, so aqueous environments are necessary for survival and transmission among mammals sharing a common environment (Adler, 2015a). Transmission of Leptospira to susceptible hosts usually occurs through contact with urine or water sources contaminated with animal urine [START_REF] Ko | Leptospira: the dawn of the molecular genetics era for an emerging zoonotic pathogen[END_REF]. Sick animals usually shed virulent leptospires during the immune phase and convalescence of the disease (asymptomatic carriers). Rodents, on the other hand, usually are resistant to disease, but can remain infected and act as continuous reservoirs, shedding infecting leptospires through urine to environment for life [START_REF] Thiermann | The Norway rat as a selective chronic carrier of Leptospira icterohaemorrhagiae[END_REF]. Most mammals can carry leptospires with no signs of disease or only mild clinical manifestations. Such subclinical infections are caused mainly (but not exclusively) by host-adapted serovars. The three main environments indicating human transmission risks are water-based, rodent-borne and livestock/pet-borne environments.

1.3.2 Human, animal, and environmental reservoirs Leptospirosis is primarily an animal disease affecting wild and domestic mammals. Acute leptospirosis in animals can present with the same clinical patterns as in humans, mostly in young animals. After the septicemic phase, once the circulating antibody titers have risen, pathogenic serovars of Leptospira usually accumulate in kidneys, inside the renal tubules; so that infected animals shed potentially infecting leptospires to the environment through their urine. It causes important losses to livestock operations (especially cattle and swine), mainly because of abortion and infertility. In dogs the disease shows at least three different pathologic syndromes: icteric syndrome, uremic syndrome and hemorrhagic syndrome [START_REF] Adler | Leptospira and leptospirosis[END_REF]. Seroprevalence in cattle may be as high as 84% and in dogs 35% [START_REF] De Freitas | Prevalence of Leptospira interrogans antibodies in free-ranging Tayassu pecari of the Southern Pantanal, Brazil, an ecosystem where wildlife and cattle interact[END_REF], Ellis et al., 1986b, Oliveira Lavinsky et al., 2012[START_REF] Schuller | Prevalence of antileptospiral serum antibodies in dogs in Ireland[END_REF]. Lower incidences have been found in other domestic animals among them sheep acting mainly as asymptomatic reservoirs [START_REF] Cerri | Leptospira interrogans serovar hardjo in the kidneys and genital tracts of naturally infected sheep[END_REF][START_REF] Ellis | Seroprevalence to Leptospira interrogans serovar hardjo in merino stud rams in South Australia[END_REF][START_REF] Lilenbaum | Identification of Leptospira spp. carriers among seroreactive goats and sheep by polymerase chain reaction[END_REF]; in horses the disease usually has a chronic course resulting in recurrent uveitis (ERU) being the main cause of equine blindness [START_REF] Halliwell | Studies on equine recurrent uveitis. II: The role of infection with Leptospira interrogans serovar pomona[END_REF][START_REF] Malalana | Equine recurrent uveitis: Human and equine perspectives[END_REF][START_REF] Wollanke | Serum and vitreous humor antibody titers in and isolation of Leptospira interrogans from horses with recurrent uveitis[END_REF]. Cats are usually resistant to infection unless a concurrent immunosuppressive disease might be affecting them, such as feline leukemia virus or feline AIDS [START_REF] Azocar-Aedo | Leptospira spp. in Domestic Cats from Different Environments: Prevalence of Antibodies and Risk Factors Associated with the Seropositivity[END_REF][START_REF] Hartmann | Leptospira Species Infection in Cats: ABCD guidelines on prevention and management[END_REF]. Outbreaks of leptospirosis have also been described in wild animals such as sea lions in California [START_REF] Avalos-Téllez | Pathogenic Leptospira serovars in free-living sea lions in the gulph of California and along the Baja California coast of Mexico[END_REF][START_REF] Colagross-Schouten | Diagnosis and seroprevalence of leptospirosis in California sea lions from coastal California[END_REF][START_REF] Prager | Asymptomatic and chronic carriage of Leptospira interrogans serovar Pomona in California sea lions (Zalophus californianus)[END_REF]. Other wild species such as rhinoceroses and wolves may suffer of acute leptospirosis, but probably only when kept in captivity [START_REF] Neiffer | Leptospira infection in two black rhinoceroses (Diceros bicornis michaeli)[END_REF]. Rodents play an important role in transmission and are considered the principal reservoirs that maintain leptospires present in environment and the main source of infection to susceptible hosts such as pigs, dogs, zoo animals and humans [START_REF] Athanazio | Rattus norvegicus as a model for persistent renal colonization by pathogenic Leptospira interrogans[END_REF][START_REF] Costa | Influence of household rat infestation on leptospira transmission in the urban slum environment[END_REF][START_REF] Haake | Leptospirosis in humans[END_REF]. De [START_REF] De Faria | Carriage of Leptospira interrogans among domestic rats from an urban setting highly endemic for leptospirosis in Brazil[END_REF] reported that up to 80.3% of Norway rats (Rattus norvegicus), a species that spread almost all over the globe, were culture positive and 68.1% were shown positive by antibody titers of at least 1:100 in a microscopic agglutination test (MAT). Other species of wild mammals have been detected as leptospiral carriers [START_REF] Andreoli | Leptospira spp. infection in wild ruminants: a survey in Central Italian Alps[END_REF][START_REF] Duncan | Leptospirosis and tularaemia in raccoons (Procyon lotor) of Larimer County[END_REF][START_REF] Pinna | Detection of anti-Leptospira antibodies in captive nonhuman primates from Salvador, Brazil[END_REF][START_REF] Scialfa | First isolation of Leptospira interrogans from Lycalopex griseus (South American gray fox) in Argentina shows new MLVA genotype[END_REF]. In France, seropositivity rates were found to range from 31% in farmed American mink, 74% in wild European mink and up to 89% in stone martens [START_REF] Moinet | Leptospirosis in free-ranging endangered European mink (Mustela lutreola) and other small carnivores (Mustelidae, Viverridae) from southwestern France[END_REF]. In the United Kingdom, seropositivity rates were 6.2% in water voles [START_REF] Gelling | Leptospirosis acquisition following the reintroduction of wildlife[END_REF]. Bats from different continents have also been shown as carriers of different Leptospira species, with a variable excretion rates (from 6 to 45%) [START_REF] Dietrich | Leptospira and Bats: Story of an emerging friendship[END_REF]. Most infected animals recover and become asymptomatic carriers for long periods of time, shedding leptospires through their urine (Della Rossa et al., 2016[START_REF] Ko | Leptospira: the dawn of the molecular genetics era for an emerging zoonotic pathogen[END_REF], Villanueva et al., 2014). Some serovars are adapted to a specific animal species, serovar Canicola to dogs [START_REF] Morrison | Canine leptospirosis: an immunopathological study of interstitial nephritis due to Leptospira canicola[END_REF], serovar Hardjo to cattle (Ellis et al., 1986b), serovars Pomona and Bratislava to pigs (Ellis et al., 1986a;[START_REF] Stalheim | Vaccination of hamsters, swine, and cattle with viable, avirulent Leptospira pomona[END_REF], serovar Pomona to horses [START_REF] Halliwell | Studies on equine recurrent uveitis. II: The role of infection with Leptospira interrogans serovar pomona[END_REF], Verma et al., 2013a), serovar Ballum to mice (Carmona Gasca et al., 2013;[START_REF] Da Silva | Preliminary Characterization of Mus musculus-Derived Pathogenic Strains of Leptospira borgpetersenii Serogroup Ballum in a Hamster Model[END_REF][START_REF] Brem | Leptospira infected rat population as probable cause of a fatal case of Weil's disease[END_REF], serovar Icterohaemorrhagiae to rats [START_REF] Thiermann | The Norway rat as a selective chronic carrier of Leptospira icterohaemorrhagiae[END_REF][START_REF] Adler | Leptospira and leptospirosis[END_REF]. Except in specific endemic settings [START_REF] Ganoza | Asymptomatic Renal Colonization of Humans in the Peruvian Amazon by Leptospira[END_REF], chronic renal carriage in humans is very rare and does not significantly contribute to the epidemiology of leptospirosis. Human to human transmission is a very rare event and humans mostly acquire the disease directly from animals (i.e., contact with urine or tissues) or more frequently indirectly (i.e., contact with urine-contaminated water) [START_REF] Koizumi | Human leptospirosis cases and the prevalence of rats harbouring Leptospira interrogans in urban areas of Tokyo, Japan[END_REF][START_REF] Ko | Leptospira: the dawn of the molecular genetics era for an emerging zoonotic pathogen[END_REF].

Incubation period

Leptospirosis has a variable incubation period depending on virulence of the infecting strain, infecting dose and the patient susceptibility [START_REF] Levett | Leptospirosis[END_REF]. Entrance of pathogenic Leptospira to susceptible hosts occurs through mucous membranes or skin abrasions [START_REF] Adler | Leptospira and leptospirosis[END_REF][START_REF] Levett | Leptospirosis[END_REF]. Leptospires reach the bloodstream and replicate in the absence of specific antibodies (leptospiremia), disseminating to many tissues [START_REF] Coutinho | Kinetics of Leptospira interrogans infection in hamsters after intradermal and subcutaneous challenge[END_REF][START_REF] Ko | Leptospira: the dawn of the molecular genetics era for an emerging zoonotic pathogen[END_REF]. This phase is called septicemic phase and typically is characterized by fever, myalgia, arthralgia, erythema and cephalgia [START_REF] Alston | Leptospiral Jaundice Among Sewer-workers[END_REF] and lasting from three to ten days. After this phase, serum antibodies become elevated and leptospires correspondingly disappear from the bloodstream [START_REF] Ko | Leptospira: the dawn of the molecular genetics era for an emerging zoonotic pathogen[END_REF][START_REF] Levett | Leptospirosis[END_REF]. Studies in animals, particularly hamsters, have revealed that most tissues are invaded, especially kidneys, where leptospires allocate into the renal tubules away from the circulating specific antibodies [START_REF] Coutinho | Kinetics of Leptospira interrogans infection in hamsters after intradermal and subcutaneous challenge[END_REF]. Leptospires are subsequently shed to the environment via urine (leptospiruria). This second phase of the disease, in which leptospires are shed, is called the immune phase and can last for long periods of time, even for life in reservoir animals [START_REF] Ko | Leptospira: the dawn of the molecular genetics era for an emerging zoonotic pathogen[END_REF]. Many of the severe symptoms of this phase of the disease may be due to immunopathology [START_REF] Fraga | Leptospirosis: Aspects of Innate Immunity, Immunopathogenesis and Immune Evasion from the Complement System[END_REF].

Period of communicability

Although very rare cases of inter-human transmission have been suspected and reported, leptospirosis is not a contagious disease. Therefore, this section discusses communicability from the animal reservoir and human susceptibility. Three main factors intervene to define the period during which sick animals, asymptomatic carriers or reservoirs may shed infecting leptospires to the environment [START_REF] Adler | Leptospira and leptospirosis[END_REF][START_REF] Holt | A model of Leptospirosis infection in an African rodent to determine risk to humans: seasonal fluctuations and the impact of rodent control[END_REF].

Serovar virulence. Virulence obviously and directly 1.

relates to the infective dose. It has been shown that Leptospira serovars vary in their level of pathogenicity. There are differences in virulence (level of pathogenicity), even among isolates of the same serovar. Some extremely virulent serovars have an experimental LD 50 of four or less bacteria (CFU) per animal in the hamster model (Sánchez, 2008;[START_REF] Watanabe | Classic dose-response and time postinoculation models for leptospira[END_REF]. In any case, the infectious dose is crucial to the severity of the disease. Even though some species-adapted serovars may cause mild or no obvious signs of disease, they may still be shed to the environment through urine. Immune status of the host. Animals that have never 2.

been in contact with a virulent Leptospira serovar, young or immunosuppressed individuals are more prone to acquire a severe clinical infection, relative to adults or immune competent individuals that develop a mild disease. Severe clinical infection may end in death of sick animals. Chronic carriers, in contrast, may shed infectious leptospires for long periods of time, even for life. Of note, leptospirosis immunity is both short lasting and serogroupspecific. People in hyperendemic areas may get leptospirosis several times, either from strains belonging to different serogroups or after postinfection immunity has declined.

The serovar -animal species relationship. It is well 3.

known that species-adapted serovars develop mild or subclinical infections in the corresponding animal species, frequently leading to chronic carriage in their animal host and chronic urinary shedding [START_REF] Levett | Leptospirosis[END_REF]. In contrast, serovars non-adapted to the infected species usually produce severe or even fatal infections, but result in short-term excretion.

Depending on these three factors, an animal may, on the one hand, become severely sick and even die from the leptospiral infection, so that the period of shedding might be short or even non-existent. On the other hand, if the infection is not so severe as to kill the patient/animal, the relapse and recovery can lead to a state of chronicity and animals then become asymptomatic carriers for long periods of time and even for life (Adler, 2015a). Numbers shed in urine are rarely reported, but are usually high, e.g. Gillespie and Ryno reported a direct microscopic count of up to 108 leptospires per milliliter shed in cow's urine [START_REF] Gillespie | Epidemiology of Leptospirosis[END_REF].

Population susceptibility

As mentioned above, leptospirosis affects more than one million people a year worldwide, and almost 60,000 fatal cases per year [START_REF] Costa | Global Morbidity and Mortality of Leptospirosis: A Systematic Review[END_REF][START_REF] Ko | Leptospira: the dawn of the molecular genetics era for an emerging zoonotic pathogen[END_REF]. Farmers and suburban slum inhabitants are at the highest risk of infection [START_REF] Costa | Influence of household rat infestation on leptospira transmission in the urban slum environment[END_REF], however there is one report suggesting that natural bodies of water in urban settings may pose higher risks of severe leptospirosis than rural ones [START_REF] Ganoza | Determining risk for severe leptospirosis by molecular analysis of environmental surface waters for pathogenic Leptospira[END_REF]. There have been reports of human leptospirosis outbreaks associated with urine contamination of water sources presenting a fatality rate of 8.6% (3 out of 35) [START_REF] Cacciapuoti | A waterborne outbreak of leptospirosis[END_REF]. Most outbreaks however are related to flooding following heavy exceptional or seasonal rainfall [START_REF] Wynwood | The emergence of Leptospira borgpetersenii serovar Arborea as the dominant infecting serovar following the summer of natural disasters in Queensland, Australia 2011[END_REF][START_REF] Smith | Leptospirosis following a major flood in Central Queensland, Australia[END_REF][START_REF] Easton | Leptospirosis in Philippine floods[END_REF][START_REF] Gaynor | Leptospirosis on Oahu: an outbreak associated with flooding of a university campus[END_REF][START_REF] Weinberger | El Niño Southern Oscillation and leptospirosis outbreaks in New Caledonia[END_REF]. However, there is at least one report where the main risk factor was the opposite, subnormal rainfall that may have contributed to the risk of exposure in military personnel exposed to contaminated water sources [START_REF] Corwin | A waterborne outbreak of leptospirosis among United States military personnel in Okinawa, Japan[END_REF]. The environmental exposure is detailed in section 2. Of note, males are infected more frequently than females, possibly reflecting both an occupational nature of the transmission and sex-related susceptibility [START_REF] Costa | Influence of household rat infestation on leptospira transmission in the urban slum environment[END_REF][START_REF] Haake | Leptospirosis in humans[END_REF][START_REF] Gomes | Sex matters: male hamsters are more susceptible to lethal infection with lower doses of pathogenic Leptospira[END_REF]. Global incidence rates are indicated in Table 1.

Population and Individual Control Measures

Vaccines

After the first isolation of Leptospira in Japan in 1914, Japanese researchers showed two years later that a phenolinactivated culture of a virulent Leptospira elicited immunity against infection in Guinea pigs [START_REF] Ido | The prophylaxis of Weil's disease (Spirochaetosis Icterohaemorrhagica)[END_REF]Adler, 2015b). To date, different methods for inactivation of leptospires for use as vaccines have been applied, these include formalin, phenol, ethanol, heat, freeze and thaw and radiation. After 100 years, the only licensed vaccines are whole Leptospira inactivated bacterins (Adler, 2015b). As leptospirosis is largely a zoonotic disease, it is important to consider animal immunization as part of a control strategy -hence the detailed discussion in this section on vaccines. Current practice should consider immunization of local animals, such as dogs, cattle and pigs.

Leptospirosis is considered a neglected disease, and the spirochete causing this disease, Leptospira, is very difficult to recover in culture. Only recently have molecular genetics tools been developed that are suitable for the study of this spirochete (Adler, 2015a;[START_REF] Evangelista | Leptospira as an emerging pathogen: a review of its biology, pathogenesis and host immune responses[END_REF][START_REF] Ko | Leptospira: the dawn of the molecular genetics era for an emerging zoonotic pathogen[END_REF]. These factors have contributed to a lag in the understanding of its virulence mechanisms, its immunogenic properties and finally a lag in the development of vaccines that may induce robust and lasting protection (Adler, 2015b;[START_REF] Evangelista | Leptospira as an emerging pathogen: a review of its biology, pathogenesis and host immune responses[END_REF].

Global coverage and future prospects

To date, available Leptospira vaccines only provide a short lasting and serogroup-specific immunity. Problems with components of the culture media and reactogenicity have also been suspected, although not demonstrated [START_REF] Benbrik | Evaluation de la tolérance de la vaccination contre Leptospira icterohaemorrhagiae chez 50 employés de canaux[END_REF]. Therefore, such bacterins have not been widely used in humans. However human bacterin vaccines have been used successfully in several regions, including China, Japan, Cuba, and Europe (Verma et al., 2013b;[START_REF] Zamora | Scale-up of the fermentation of Leptospira interrogans canicola canicola for the production of vax-SPIRAL vaccine[END_REF], notably to prevent occupational leptospirosis in sewage-workers [START_REF] De Serres | Need for vaccination of sewer workers against leptospirosis and hepatitis A[END_REF][START_REF] Nardone | Risk factors for leptospirosis in metropolitan France: results of a national case-control study, 1999-2000[END_REF]. Importantly, animal vaccination might still prove useful in reducing leptospirosis burden in humans, by decreasing the environmental contamination. In this regard, dog vaccination is routinely performed in a number of countries, aiming at protecting both the dog and its owner [START_REF] Gay | Though not reservoirs, dogs might transmit Leptospira in New Caledonia[END_REF]. Livestock vaccination is widely implemented in New Zealand and correlated with a significant decrease in human cases [START_REF] Thornley | Changing epidemiology of human leptospirosis in New Zealand[END_REF]. Further details on vaccines are presented in Appendix A.

Hygiene measures

Currently, implementing rodent control through improved sanitation and food residuals management seems the most effective measures to limit leptospirosis. Farmers and other professionals in occupations at risk should use protective gear (apron, gloves, boots and protective mask), e.g. in abattoirs or when using high pressure water (for cleaning) to avoid infection by aerosolized animal urine. Additionally, animal vaccination may reduce leptospiral shedding [START_REF] Subharat | Growth response and shedding of Leptospira spp. in urine following vaccination for leptospirosis in young farmed deer[END_REF]. For workers exposed to municipal wastewaters (e.g. sewage workers), vaccination, even in developed regions would seem appropriate for risk management [START_REF] De Serres | Need for vaccination of sewer workers against leptospirosis and hepatitis A[END_REF], however vaccines for humans are available only in a few countries.

Environmental Occurrence and Persistence

Studies of virulent Leptospira in the environment are very limited, mainly because it is difficult to isolate these spirochetes from surface waters or soil without contamination with saprophytic Leptospira. However, there is a growing interest in understanding the lifestyle and survival capabilities of virulent Leptospira in the environment. In addition, genomic studies suggest that virulent Leptospira evolved from an environmental (freeliving) Leptospira present in water or mud, similarly to the contemporary members of the saprophytic cluster (such as L. biflexa) [START_REF] Saito | Comparative analysis of Leptospira strains isolated from environmental soil and water in the Philippines and Japan[END_REF]Thibeaux et al., 2018b;Thibeaux et al., 2018a). There seems to be some evolutionary variation within virulent leptospires; some species (such as L. interrogans) have retained genes associated to environmental survival whereas other virulent members (such as L. borgpetersenii) seem to have lost these genes. These findings have been corroborated by in vitro experiments showing that L. interrogans survives better in distilled water than L. borgpetersenii [START_REF] Bulach | Genome reduction in Leptospira borgpetersenii reflects limited transmission potential[END_REF][START_REF] Picardeau | Genome Sequence of the Saprophyte Leptospira biflexa Provides Insights into the Evolution of Leptospira and the Pathogenesis of Leptospirosis[END_REF], which may imply that L. borgpetersenii is not transmitted by water but rather relies on direct host-to-host transmission [START_REF] Bulach | Genome reduction in Leptospira borgpetersenii reflects limited transmission potential[END_REF].

Leptospiral survival in water is bolstered by viscous material, such as agar, which possibly translates to biofilm in natural environments. Some strains of virulent Leptospira are able to produce biofilms when incubated in fresh water, even in the absence of nutrients [START_REF] Barragan | Interactions of Leptospira with Environmental Bacteria from Surface Water[END_REF][START_REF] Ristow | Biofilm formation by saprophytic and pathogenic leptospires[END_REF]. Furthermore L. interrogans (pathogenic cluster) has been found in nature associated with biofilms formed by environmental bacteria (Vinod Kumar et al., 2015a). Some virulent Leptospira are able to produce biofilm when incubated in distilled water (low nutrient conditions) and appear sense the presence of nutrients, such as fatty acids, which triggers release of leptospiral cells from the biofilm [START_REF] Barragan | Interactions of Leptospira with Environmental Bacteria from Surface Water[END_REF]. It has been observed that biofilm-borne leptospiral cells display a five-to six-fold increase in antibiotic tolerance (Vinod Kumar et al., 2016a). It has also been speculated that biofilms protect pathogenic Leptospira against other toxic compounds and stressors in the environment. Taken together, Leptospira survival in the environment seems to be favored by biofilm formation, which could contribute to disease transmission among risk groups such as sewage and agricultural workers (Della [START_REF] Rossa | Environmental factors and public health policy associated with human and rodent infection by leptospirosis: a land cover-based study in Nan province, Thailand[END_REF][START_REF] Ganoza | Determining risk for severe leptospirosis by molecular analysis of environmental surface waters for pathogenic Leptospira[END_REF][START_REF] Koizumi | Human leptospirosis cases and the prevalence of rats harbouring Leptospira interrogans in urban areas of Tokyo, Japan[END_REF][START_REF] Trueba | Cell aggregation: a mechanism of pathogenic Leptospira to survive in fresh water[END_REF].

Soil may also play a role in environmental survival. Some researchers have shown that Leptospira survived in the wet soil on dry days and appeared in surface water on rainy days, suggesting that soil could be a reservoir for leptospires in the environment [START_REF] Saito | Comparative analysis of Leptospira strains isolated from environmental soil and water in the Philippines and Japan[END_REF][START_REF] Thibeaux | Seeking the environmental source of Leptospirosis reveals durable bacterial viability in river soils[END_REF]. Even though leptospires are considered fragile spirochetes, studies have shown that they can survive and even maintain their virulence for up to 20 months despite unfavorable conditions, such as cold and nutrient-poor acidic waters [START_REF] Andre-Fontaine | Waterborne Leptospirosis: Survival and Preservation of the Virulence of Pathogenic Leptospira spp. in Fresh Water[END_REF]. However, there is limited knowledge about possible differences in the nutritional requirements of saprophytic and virulent species and about ability of virulent leptospires to multiply in surface water or soils. General data on the occurrence of leptospires in the environment is presented in Tables 2345. a Liquid and solid EMJH supplemented with 5-Fluoro-Uracil (100 µg/ml), rabbit serum 1%, Amphotericine B (50µg/ml), Sulphatiazole (50 mg/L), neomycin sulphate (5 mg/L), actidione (0.5 mg/L) and incubated at 29°C. Identification by serological typing.

Detection Methods

Leptospires might be detected by either culture and isolation or using molecular techniques like (quantitative) PCR. Leptospira spp. are fastidious slow-growing organisms with very specific nutritional requirements. However, as for most bacterial species, initial work used classical, culturebased isolation procedures. Although several different culture media have been developed and used for Leptospira, most studies have used the oleic acid -albumin culture medium Ellinghausen-McCullough-Johnson-Harris (EMJH) or the rabbit serum-enriched Korthof's enrichments; as liquid or semi-solid media for at least initial culture, thus preventing quantification. Since the mid 1960s, the control of contaminants has been achieved by including 5-Fluoro-Uracil into the culture medium [START_REF] Johnson | 5-Fluorouracil as a Selective Agent for Growth of Leptospirae[END_REF]. More recently, a combination of selective antimicrobial agents as proposed by [START_REF] Chakraborty | A novel combination of selective agents for isolation of Leptospira species[END_REF] has increasingly been used (sulfamethoxazole, 40 μg/mL; trimethoprim, 20 μg/mL; amphotericin B, 5 μg/mL; fosfomycin, 400 μg/mL; and 5-fluorouracil, 100 μg/mL). Whether these culture media and selective agents favor the growth of all Leptospira species and strains is still unknown, and the isolation process most likely results in the loss of Leptospira diversity. In addition, although viable but non-culturable (VBNC) Leptospira have not been reported, the ability of different strains to grow in vitro in culture media is still very poorly established.

As with any culture-based isolation technique, fast-growing saprophytic species are most frequently isolated and may jeopardize the detection and isolation of slower-growing virulent leptospires. Historically, to gain insight into pathogenic Leptospira from waters and soils, direct inoculation of environmental samples into susceptible animal models have been used. Pathogenic leptospires that induce infection and disease in such test animals and cultures from their blood or target organs allows for the recovery of pure cultures. Examples are provided in the following section 2.2, pathogenic leptospires were isolated from recreational waters in the USA after inoculation into guinea pigs [START_REF] Diesch | Isolation of pathogenic leptospires from waters used for recreation[END_REF].

A number of pathogenic leptospires were similarly recovered from soils and waters in Malaysia after inoculation of hamsters [START_REF] Baker | Pathogenic Leptospira in Malaysian surface waters. I. A method of survey for Leptospira in natural waters and soils[END_REF]. This technique used many susceptible animals (hamsters or guinea pigs), and is currently unacceptable from an ethical perspective. Of note, this technique does not allow a quantitative assessment, since the pathogenic leptospires are amplified or lost in the test animals by immune clearance, depending both on their own virulence and their initial concentration in the sample studied (minimal infective dose). Animals might also die from another infection that may mask a virulent leptospire. To summarize, this technique has an excellent specificity if Leptospira isolation is successful, but may suffer from a low sensitivity presumably leading to a number of false negative results. Consequently, the proportion of positive samples observed with this animal-enrichment technique are expected to underestimate the true occurrence.

Once the leptospires are isolated, there is no easy and specific phenotypic technique for their identification, even at the species level. This further complicated the early studies of Leptospira in the environment, but modern molecular methods are now reducing this problem. The International Leptospirosis Society regularly produces a position statement on the speciation of Leptospiral isolates, which is publicly available (https://leptosociety.org/resources). The most recent recommendation is to use Whole Genome Sequencing techniques and genomic comparisons for speciation.

Culture from environmental samples

There is no standard protocol for culturing virulent species of Leptospira from bodies of fresh water or soil. Furthermore, there are only a few reports describing the isolation of virulent Leptospira from environmental samples; as discussed above, the main difficulty in the isolation of these species is the overgrowth of environmental bacteria or the fast-growing saprophytic Leptospira [START_REF] Barragan | Interactions of Leptospira with Environmental Bacteria from Surface Water[END_REF][START_REF] Benacer | Pathogenic and Saprophytic Leptospira Species in Water and Soils from Selected Urban Sites in Peninsular Malaysia[END_REF][START_REF] Saito | Comparative analysis of Leptospira strains isolated from environmental soil and water in the Philippines and Japan[END_REF]. Culture-based protocols include the use EMJH (Ellinghausen and McCullough modified by Johnson and Harris) culture medium containing cocktails of antibacterial and anti-fungal compounds such as sulfamethoxazole, trimethoprim, amphotericin B, fosfomycin, and 5-fluorouracil [START_REF] Benacer | Pathogenic and Saprophytic Leptospira Species in Water and Soils from Selected Urban Sites in Peninsular Malaysia[END_REF][START_REF] Saito | The usefulness of semi-solid medium in the isolation of highly virulent Leptospira strains from wild rats in an urban area of Fukuoka, Japan[END_REF][START_REF] Saito | Comparative analysis of Leptospira strains isolated from environmental soil and water in the Philippines and Japan[END_REF]. Alternatively bacterial contamination in water samples can be reduced by utilizing 0.22 µm filtrate before enriching in EMJH medium, then isolating colonies on solid medium [START_REF] Saito | The usefulness of semi-solid medium in the isolation of highly virulent Leptospira strains from wild rats in an urban area of Fukuoka, Japan[END_REF][START_REF] Wuthiekanun | Rapid isolation and susceptibility testing of Leptospira spp. using a new solid medium, LVW agar[END_REF].

Moleular based methods: amplification of leptospiral DNA from environmental samples

Due to difficulties in culturing Leptospira from environmental samples, numerous PCR-based techniques have been developed that mainly target genes only present in pathogenic Leptospira, such as lipL32 [START_REF] Muñoz-Zanzi | Leptospira Contamination in Household and Environmental Water in Rural Communities in Southern Chile[END_REF]Vinod Kumar et al., 2016b;[START_REF] Aviat | Synthetic peptide issued from Hap1/LipL32 for new early serodiagnosis of human leptospirosis[END_REF] or pathogenic specific sequences of genes, such as flaB [START_REF] Villanueva | Leptospira-rat-human relationship in Luzon, Philippines[END_REF], secY (Perez and Goarant, 2010;[START_REF] Guernier | Advances and challenges in barcoding pathogenic and environmental Leptospira[END_REF] and the 16S rRNA gene [START_REF] Ganoza | Determining risk for severe leptospirosis by molecular analysis of environmental surface waters for pathogenic Leptospira[END_REF]. Because of sequence polymorphisms, PCR targeting lipL32 or secY fail to detect virulent Leptospira of the intermediate cluster (e.g. L. fainei, L. broomii, L. inadai, L wolffii, L. licerasiae), which are commonly found in water [START_REF] Ganoza | Determining risk for severe leptospirosis by molecular analysis of environmental surface waters for pathogenic Leptospira[END_REF][START_REF] Thaipadungpanit | Leptospira Species in Floodwater during the 2011 Floods in the Bangkok Metropolitan Region, Thailand[END_REF].

A second problem with PCR methods used to date is that detection of genomic DNA sequences provides no indication of the viability of bacteria in water or other matrices. In addition to the possible use of PMA or EMA to reduce cell membrane compromised or free DNA detections [START_REF] Thibeaux | Seeking the environmental source of Leptospirosis reveals durable bacterial viability in river soils[END_REF][START_REF] Soupé-Gilbert | Continuous excretion of Leptospira borgpetersenii Ballum in mice assessed by viability-quantitative PCR[END_REF], targeting pre-RNA appears to be an alternative with possibly greater precision in detecting viable bacteria [START_REF] Cangelosi | Prospects for applying virulence factor-activity relationships (VFAR) to emerging waterborne pathogens[END_REF][START_REF] Gedalanga | Development of a quantitative PCR method to differentiate between viable and nonviable bacteria in environmental water samples[END_REF]. While yet to be reported for Leptospira, targeting pre-RNA before and after media stimulation and assaying by qRT-PCR to identify differences in the preRNA copies present should correspond to viable Leptospira (Cangelosi, 2018). Most recently, detailed procedures for the molecular detection of pathogenic leptospires have been published, which will probably trigger further studies on leptospires in the environment [START_REF] Beigel | Leptospira: Molecular Detection of Pathogenic Species in Natural Sources[END_REF][START_REF] Riediger | An Optimized Method for Quantification of Pathogenic Leptospira in Environmental Water Samples[END_REF]Thibeaux et al., 2018a). There has also been increased use of high throughput sequencing technologies. For example, 16S rRNA amplicon sequencing yielded evidence of the presence of Leptospira in various water sources [START_REF] Huang | A comprehensive insight into bacterial virulence in drinking water using 454 pyrosequencing and Illumina high-throughput sequencing[END_REF][START_REF] Zhang | Benefits of Genomic Insights and CRISPR-Cas Signatures to Monitor Potential Pathogens across Drinking Water Production and Distribution Systems[END_REF][START_REF] Escobedo-Hinojosa | Analysis of bacterial metagenomes from the Southwestern Gulf of Mexico for pathogens detection[END_REF]. Studies using such socalled next generation DNA sequencing approaches will likely provide improved information on Leptospira ecology in the coming years.

Data on Occurrence

Excreta in the environment

Pathogenic Leptospira live and multiply in the proximal kidney tubules of reservoir animals, which are frequently asymptomatic, and are then shed through the urine. Thus, they reach soils and waters in the environment, which are the sources of most human infection. However, very little is known on the actual numbers of leptospires that are shed by reservoir animals. A detailed analysis of the kinetics of excretion in mice shows that excretion increases with age, reaching 3.0 x10 7 /ml of urine and suggesting that older rodents excrete larger amounts of leptospires [START_REF] Soupé-Gilbert | Continuous excretion of Leptospira borgpetersenii Ballum in mice assessed by viability-quantitative PCR[END_REF]. A meta-analysis indicated that reservoir rats excrete the greatest concentration of leptospires (5.7x10 6 per mL of urine), but large mammals produce the greatest quantities (up to 10 9 leptospires per day) because of the larger volume of urine [START_REF] Barragan | Meta-analysis to estimate the load of Leptospira excreted in urine: beyond rats as important sources of transmission in low-income rural communities[END_REF]. This meta-analysis suggested that humans could shed approximately 10 6 leptospires per day. However, except in very specific situations, humans usually do not become chronic carriers and only excrete leptospires for a couple of days to weeks [START_REF] Levett | Leptospirosis[END_REF]. Therefore, human excreta are usually not considered to play an epidemiological role in transmission of leptospirosis.

Sewage

Leptospirosis has been historically associated with sewage and is still an occupational hazard in sewerworkers. Pathogenic leptospires were isolated from sewage or sewage slime very early [START_REF] Alston | Leptospiral Jaundice Among Sewer-workers[END_REF][START_REF] Coghlan | A new serovar mogdeni of serogroup Tarassovi of Leptospira interrogans isolated from a sewage plant in England[END_REF][START_REF] Jones | The occurrence and significance to animal health of Leptospira, Mycobacterium, Escherichia coli, Brucella abortus and Bacillus anthracis in sewage and sewage sludges[END_REF]. Using molecular techniques, pathogenic leptospires were evidenced from sewage in the Peruvian Amazon [START_REF] Ganoza | Determining risk for severe leptospirosis by molecular analysis of environmental surface waters for pathogenic Leptospira[END_REF] and from a wastewater sample in Colombia [START_REF] Ensuncho-Hoyos | Epidemiology behavior of leptospirosis in Cienaga de Oro, Cordoba (Colombia)[END_REF]. In Brazilian slums, open sewers are considered as a major source of human leptospirosis, yet may be more a function of rat urine presence than human sewage. A recent study in an urban slum in Salvador, Brazil, demonstrated the presence of pathogenic leptospires in more than one third of sewage water samples, with a concentration that range 2.0x10 3 to 1.7x10 6 /100 mL. The highest concentration were reported during the rainy season and at sampling sites nearest the bottom of the valley and thus most vulnerable to flood waters [START_REF] Casanovas-Massana | Spatial and temporal dynamics of pathogenic Leptospira in surface waters from the urban slum environment[END_REF].

Manure

Due to the huge quantities of leptospires potentially excreted in the urine by large mammals, manure can be a major source of contamination. Direct detection in or isolation of pathogenic leptospires from manure is a major challenge, because of the enormous bacterial burden and the difficulties of selectively growing leptospires, as well as the major concern of PCR inhibitors preventing a relevant use of molecular detection. Cattle manure was shown to be a source of pathogenic leptospires in field conditions [START_REF] Gillespie | Epidemiology of Leptospirosis[END_REF]. A study that modeled the shedding of leptospires in cattle manure found they were resilient in an oxidation ditch designed for nutrient removal, persisting for upwards of two months [START_REF] Diesch | Survival of leptospires in cattle manure[END_REF]. More recently, a change in the management of livestock manure in Korea was associated with a significant decrease in leptospirosis incidence [START_REF] Ryu | The impact of Livestock Manure Control Policy on human leptospirosis in Republic of Korea using interrupted time series analysis[END_REF].

Surface waters

As described above (see section 2.1), studies using only culture-based methods mostly detected saprophytic leptospires, which are normal inhabitants of water and soils and frequently overgrow virulent leptospires in culture. Using inoculation to susceptible animals (guinea pigs or hamsters), pathogenic leptospires have been isolated from both temperate and tropical surface waters [START_REF] Diesch | Isolation of pathogenic leptospires from waters used for recreation[END_REF]Crawford Jr et al., 1969;[START_REF] Baker | Pathogenic Leptospira in Malaysian surface waters. I. A method of survey for Leptospira in natural waters and soils[END_REF][START_REF] Alexander | Pathogenic Leptospiras Isolated from Malaysian Surface Waters[END_REF][START_REF] Jackson | Outbreak of leptospirosis associated with swimming[END_REF]. Molecular detection has been used since the late 1990s and notably allowed some DNA from pathogenic Leptospira to be detected from lake water associated with a recreational outbreak of leptospirosis after a triathlon in Illinois, USA [START_REF] Morgan | Outbreak of leptospirosis among triathlon participants and community residents in Springfield, Illinois[END_REF]. Later molecular methods detected pathogenic leptospires from 23% of various surface water sources in Thailand [START_REF] Tansuphasiri | Duplex PCR-hybridization based detection of pathogenic Leptospira in environmental water samples obtained from endemic areas in northeast region of Thailand[END_REF]. Of note, classical PCR cannot provide direct quantitative information about the leptospires, but both studies cited above suggest low concentrations. The use of quantitative real time PCR (qPCR) does enable direct quantitative estimates of concentrations of leptospires in water sources. Concentrations in the range one to seventeen thousand leptospires per mL (including both pathogenic and intermediate strains) were measured in various surface water sources in Peru, notably showing both higher frequency and higher concentrations in urban (especially in a market area) than in rural area [START_REF] Ganoza | Determining risk for severe leptospirosis by molecular analysis of environmental surface waters for pathogenic Leptospira[END_REF]. In Hawaiian streams, Leptospira were detected in 87 of 88 water samples (98.8%), with concentrations from 5 to 10 4 genomes / 100mL (approx. 1 to 2.0x10 3 leptospires / 100 mL), with a strong correlation with turbidity. Further sequencing of the PCR product evidenced only species from the intermediate cluster [START_REF] Viau | Bacterial pathogens in Hawaiian coastal streams--associations with fecal indicators, land cover, and water quality[END_REF]. In a temperate environment near Lyon, France, pathogenic Leptospira were detected in 3 of 47 (6.38%) water samples collected from ponds with concentrations in the range 10 5 to 10 6 / 100 mL [START_REF] Vein | Adaptation of a real-time PCR method for the detection and quantification of pathogenic leptospires in environmental water[END_REF]. In the temperate region of Los Rios in South-Central Chile, pathogenic leptospires evidenced by PCR were more common in standing waters (19.3% of puddles and 45.5% of open containers) than in running waters (3.9% in canals or rivers). The overall detection rate was 77 out of 570 (13.5%) water samples, revealing the ubiquity of pathogenic Leptospira in surface waters in this temperate region [START_REF] Muñoz-Zanzi | Leptospira Contamination in Household and Environmental Water in Rural Communities in Southern Chile[END_REF]. Particular attention should be paid to floodwaters, which are known to be responsible for numerous leptospirosis infections and outbreaks in humans. Although floodwaters have rarely been studied [START_REF] Thaipadungpanit | Leptospira Species in Floodwater during the 2011 Floods in the Bangkok Metropolitan Region, Thailand[END_REF][START_REF] Chaturongkasumrit | Microbiological evaluation of water during the 2011 flood crisis in Thailand[END_REF], they should be considered as periods of increased leptospirosis risk, as shown by the high numbers of leptospirosis outbreaks triggered by floods [START_REF] Smith | Leptospirosis following a major flood in Central Queensland, Australia[END_REF][START_REF] Park | Brief report: Leptospirosis after flooding of a university campus--Hawaii, 2004[END_REF][START_REF] Matono | Imported Flood-Related Leptospirosis From Palau: Awareness of Risk Factors Leads to Early Treatment[END_REF][START_REF] Gaynor | Leptospirosis on Oahu: an outbreak associated with flooding of a university campus[END_REF][START_REF] Easton | Leptospirosis in Philippine floods[END_REF] (Table 2-5).

Groundwater

Few studies have investigated leptospires in ground water. Recent 16S rRNA gene amplicon studies confirm a greater abundance of Leptospira in surface than in underground waters [START_REF] Delafont | Environmental factors shaping cultured free-living amoebae and their associated bacterial community within drinking water network[END_REF]. Pathogenic Leptospira were reported from underground water sources in Thailand, but the study did not provide hydrologic details about the water sources investigated [START_REF] Kurilung | Molecular detection and isolation of pathogenic Leptospira from asymptomatic humans, domestic animals and water sources in Nan province, a rural area of Thailand[END_REF].

Drinking water

Leptospira have been detected in drinking water samples in several studies. Saprophytic leptospires are normal inhabitants of freshwater, which do not involve any sanitary risk. However, there has also been evidence of pathogenic leptospires from drinking water for animals [START_REF] Calderon | Leptospirosis in pigs, dogs, rodents, humans, and water in an area of the Colombian tropics[END_REF][START_REF] Muñoz-Zanzi | Leptospira Contamination in Household and Environmental Water in Rural Communities in Southern Chile[END_REF][START_REF] Mason | Distribution and Diversity of Pathogenic Leptospira Species in Peri-domestic Surface Waters from South Central Chile[END_REF][START_REF] Kurilung | Molecular detection and isolation of pathogenic Leptospira from asymptomatic humans, domestic animals and water sources in Nan province, a rural area of Thailand[END_REF], but also for humans [START_REF] Muñoz-Zanzi | Leptospira Contamination in Household and Environmental Water in Rural Communities in Southern Chile[END_REF][START_REF] Mason | Distribution and Diversity of Pathogenic Leptospira Species in Peri-domestic Surface Waters from South Central Chile[END_REF]. Very interestingly, a shotgun metagenomics study retrieved a Leptospira spp. draft genome from chlorinated drinking water, suggesting an ability to resist some degree of chlorination [START_REF] Zhang | Benefits of Genomic Insights and CRISPR-Cas Signatures to Monitor Potential Pathogens across Drinking Water Production and Distribution Systems[END_REF]. Similarly, a 16S rRNA gene amplicon study found Leptospira spp. sequence reads in a drinking water network, both right after chlorination (initial concentration of 0.6-0.8 mg/L free chlorine) and in final tapwater (0.1 mg/L residual chlorine); in this study, a higher relative abundance of Leptospira spp. sequence reads in tap water compared to initial water further supports the hypothesis of resistance to chlorination [START_REF] Huang | A comprehensive insight into bacterial virulence in drinking water using 454 pyrosequencing and Illumina high-throughput sequencing[END_REF]. Whether this resistance is related to Leptospira ability to form biofilms (discussed above in section 2) remains to be determined.

Seawater

Leptospires are thought to survive poorly in seawater. However, there is limited evidence of some degree of saltwater tolerance by some Leptospira strains. One study reported the isolation of a virulent leptospire that was apparently tolerant to seawater (Grune [START_REF] Loffler | Isolation of a Seawater Tolerant Leptospira spp. from a Southern Right Whale (Eubalaena australis)[END_REF]. Another study, using 16S rRNA gene amplicon, reported Leptospira in marine waters and soils in the Gulf of Mexico [START_REF] Escobedo-Hinojosa | Analysis of bacterial metagenomes from the Southwestern Gulf of Mexico for pathogens detection[END_REF]. On the other hand, leptospirosis cases observed in marine mammals could be due to coastal contamination by virulent leptospires arising from terrestrial runoff and contamination of coastal marine environments [START_REF] Bogomolni | Victims or vectors: a survey of marine vertebrate zoonoses from coastal waters of the Northwest Atlantic[END_REF][START_REF] Cameron | Detection of Pathogenic Leptospira in Pinniped Populations via PCR Identifies a Source of Transmission for Zoonotic Leptospirosis in the Marine Environment[END_REF][START_REF] Prager | Asymptomatic and chronic carriage of Leptospira interrogans serovar Pomona in California sea lions (Zalophus californianus)[END_REF].

Sludge

Leptospires have been reported from sewage and sewage sludge since the 1930s [START_REF] Alston | Leptospiral Jaundice Among Sewer-workers[END_REF][START_REF] Jones | The occurrence and significance to animal health of Leptospira, Mycobacterium, Escherichia coli, Brucella abortus and Bacillus anthracis in sewage and sewage sludges[END_REF][START_REF] Coghlan | A new serovar mogdeni of serogroup Tarassovi of Leptospira interrogans isolated from a sewage plant in England[END_REF], yet there has been little interest generally in such reports because human sewage does not appear to be a significant source of human disease.

2.2.9 Soil [START_REF] Noguchi | The Survival of Leptospira (Spirochaeta) Icterohaemorrhagiae in Nature; Observations Concerning Microchemical Reactions and Intermediary Hosts[END_REF] appears to be one of the first to report soils as a possible environment where Leptospira survive. However, because of the technical challenges of studying delicate slow-growing organisms in soils, this environmental compartment has been poorly studied. Using animal inoculation, pathogenic Leptospira were recovered from Malaysian soil washings in the early 1960s. The authors noted a higher isolation frequency from soil washings than from waters [START_REF] Baker | Pathogenic Leptospira in Malaysian surface waters. I. A method of survey for Leptospira in natural waters and soils[END_REF]. Quite similarly, a study in Minnesota, which failed to isolate virulent strains, noted that isolation of leptospires was more frequent from soils than from adjacent waters [START_REF] Henry | Distribution of the genus Leptospira in soil and water[END_REF]. Similarly in Hawaii, leptospires were isolated from 7 of 13 water samples, but from all 16 soil samples examined (Wilson and Fujioka, 1995). In New Caledonia where leptospirosis is endemic, a source tracking study used viability-PCR and detected viable pathogenic leptospires in river soils weeks after the contamination events [START_REF] Thibeaux | Seeking the environmental source of Leptospirosis reveals durable bacterial viability in river soils[END_REF]. A combination of selective agents was proven to be very effective for the culture and isolation of Leptospira from environmental samples [START_REF] Chakraborty | A novel combination of selective agents for isolation of Leptospira species[END_REF]. Using this selection techniques, a great biodiversity of leptospires were isolated from tropical soils in New Caledonia, supporting the hypothesis that soils may be the original habitat of the genus Leptospira and a possible environmental reservoir of pathogenic strains (Thibeaux et al., 2018b;Thibeaux et al., 2018a). This hypothesis is also supported by other findings, notably the positive correlation between Leptospira concentration and turbidity (Viau and Boehm, 2011) or the role of floods in triggering leptospirosis outbreaks, as presented above.

Irrigation water and on crops

Irrigation waters are mostly surface waters, though groundwater is sometimes used. Most of the risks associated with irrigation waters should be evaluated by knowledge acquired from the water source considered and described in the corresponding section. Very few studies have considered Leptospira irrigation water.

There is both direct and indirect evidence of a high risk of leptospirosis in humans that rely on irrigated crops. In Korea, pathogenic leptospires were isolated from the water of a rice paddy field identified as a source of a leptospirosis outbreak [START_REF] Kim | Leptospirosis: a newly identified disease in Korea[END_REF], providing direct evidence. Indirect evidence relies on the epidemiological association of human leptospirosis with activity in rice paddy fields [START_REF] Kim | Leptospirosis: a newly identified disease in Korea[END_REF][START_REF] Ryu | The viability of leptospires in the summer paddy water[END_REF][START_REF] Ivanova | Leptospira and Rodents in Cambodia: Environmental Determinants of Infection[END_REF], but also other irrigated crops, such as irrigated taro fields [START_REF] Massenet | An unprecedented high incidence of leptospirosis in Futuna, South Pacific, 2004 -2014, evidenced by retrospective analysis of surveillance data[END_REF].

Crops are not considered to represent a direct risk for leptospirosis, but improper storage can lead to rodent population growth, constituting an indirect risk of leptospirosis via increased excretion of urine.

Fish and shellfish

Leptospira isolates have historically been recovered from a number of cold-blooded animals, notably amphibians and reptiles [START_REF] Andrews | Leptospiral Agglutinins in Sera from Southern Illinois Herpetofauna[END_REF][START_REF] Minette | Leptospirosis in poikilothermic vertebrates. A review[END_REF]. More recently, molecular detection suggested Leptospira carriage in the kidneys of some reptiles (Jobbins and Alexander, 2015). Positive serology was sometimes reported from freshwater fishes [START_REF] Mgode | Leptospira Serovars for Diagnosis of Leptospirosis in Humans and Animals in Africa: Common Leptospira Isolates and Reservoir Hosts[END_REF], but this finding is considered anecdotal and does not mean that the seropositive fishes had the ability to chronically carry and shed live virulent leptospires. Current scientific consensus is that cold-blooded vertebrates and invertebrates are not involved as reservoirs of humanpathogenic leptospires, and they have not been reported as possible vehicles of human contaminations. On the other hand, freshwater fish farming might pose an occupational risk for leptospirosis, through extensive and prolonged contact with freshwater, as well as through the possible presence of rodents at feed stock facilities.

Air

Leptospirosis is not an airborne infection and is only exceptionally acquired through the respiratory system. Aerosols from infectious sources (contaminated water sources, mammal urines, urine-humid beds of rodents in animal facilities) can occasionally induce leptospirosis through the mucous membranes of the respiratory tract [START_REF] Who | WHO recommended standards and strategies for surveillance prevention and control of communicable diseases: Leptospirosis. WHO recommended standards and strategies for surveillance prevention and control of communicable diseases. WHO/CDS/CSR/ISR/99[END_REF][START_REF] Levett | Leptospirosis[END_REF]. Leptospires were isolated from an air-conditioning plant in New Zealand however, the strains isolated did not demonstrate virulence in susceptible animal models, suggesting that they were not pathogenic leptospires [START_REF] Ris | The isolation of leptospires from an air-conditioning plant[END_REF].

Persistence

Environmental survival and persistence of virulent Leptospira was recognized very early on as key to leptospirosis epidemiology [START_REF] Noguchi | The Survival of Leptospira (Spirochaeta) Icterohaemorrhagiae in Nature; Observations Concerning Microchemical Reactions and Intermediary Hosts[END_REF]. Virulent leptospires are thought to multiply in animal hosts and only survive in the environment. However, because of the technical difficulties involved in culturing and identifying Leptospira in complex environmental samples, limited information on environmental Leptospira survival has been published. Most studies have used microcosms in laboratory experiments to study the survival of pathogenic leptospires under different physico-chemical conditions. In general, limited quantitative data was identified for Leptospira persistence or survival in excreta or environmental matrices. Nonetheless, factors likely to increase persistence include coaggregation with different bacteria within biofilms (Vinod Kumar et al., 2015b). Hence, pathogenic Leptospira may persist for months in surface waters [START_REF] Barragan | Interactions of Leptospira with Environmental Bacteria from Surface Water[END_REF].

One century ago, [START_REF] Noguchi | The Survival of Leptospira (Spirochaeta) Icterohaemorrhagiae in Nature; Observations Concerning Microchemical Reactions and Intermediary Hosts[END_REF] initially demonstrated both the survival and virulence of pathogenic leptospires for up to one week in drinking water. Later in a more complex experimental design, the influence of temperature and pH were studied, showing a longer survival at neutral to slightly alkaline pH and an optimal temperature around 25-27°C, with temperatures above 60°C killing leptospires in a few seconds [START_REF] Chang | Studies on Leptospira icterohaemorrhagiae; survival in water and sewage; destruction in water by halogen compounds, synthetic detergents, and heat[END_REF]. This same study showed survival of 100 days with the addition of 1% horse serum in sterile tap water. Based on inoculation studies using susceptible animal hosts, the survival and infectivity was found to be maintained in soils for more than 40 days [START_REF] Smith | Observations on the survival of Leptospira australis A in soil and water[END_REF]. Such evidence, as well that from other as other studies, has built the case that leptospires can survive and remain infective for weeks to months in a favorable environment. However, it must also be noted that bacterial survival is not only environment-dependent, but also strain-dependent and different pathogenic Leptospira strains have shown different survival capabilities under the same environmental conditions [START_REF] Smith | The effect of pH on the survival of leptospires in water[END_REF].

The longest survival reported in nutrient-deprived conditions was reported from an experiment in a mineral bottled water held at 30°C, where a Leptospira interrogans serogroup Icterohaemorrhagiae survived and even induced a lethal disease in gerbils after 593 days [START_REF] Andre-Fontaine | Waterborne Leptospirosis: Survival and Preservation of the Virulence of Pathogenic Leptospira spp. in Fresh Water[END_REF].

In natural environments, the isolation of an identical strain five months apart in the same rain puddle suggested its ability to survive in wet soil between rain events [START_REF] Saito | Comparative analysis of Leptospira strains isolated from environmental soil and water in the Philippines and Japan[END_REF]. Similarly, using viability-PCR and genotyping, the Leptospira interrogans strain involved in human infections was shown to persist in soils from river edges and sediments where human infections had occurred 9 weeks earlier [START_REF] Thibeaux | Seeking the environmental source of Leptospirosis reveals durable bacterial viability in river soils[END_REF]. Although repeat contamination of natural areas is possible, there is strong evidence that pathogenic Leptospira not only survive, but also remain infective for weeks to months in wet soils and freshwater ecosystems. Table 6 shows early studies done on persistence more recent data are not available.

Reductions by Sanitation Management

Very limited data exists on the specific removal efficiencies of leptospires via sanitation management and traditional wastewater treatment processes. Nonetheless, general disinfection methods and physical removal techniques would be considered necessary for the control of Leptospirosis. Adequate surrogates for Leptospira removal in engineered systems have not been identified, but traditional fecal indicators (i.e., Escherichia coli and Enterococci) have recently been used to assess environmental risk during a leptospirosis outbreak in Israel [START_REF] Dadon | Outbreak of human leptospirosis linked to contaminated water bodies in Northern Israel[END_REF]. Sports events in apparently pristine surface waters have been frequently linked to leptospirosis outbreaks [START_REF] Sejvar | Leptospirosis in "Eco-Challenge" athletes, Malaysian Borneo[END_REF], suggesting that coliforms and E. coli counts may not be a good indicator of leptospiral risk [START_REF] Morgan | Outbreak of leptospirosis among triathlon participants and community residents in Springfield, Illinois[END_REF]. Therefore, whether they may be reasonable substitutes for evaluating removal techniques remains to be determined. Campylobacter spp. could also serve as a surrogate due to it also being a Gram-negative bacterium with a similarly thin and elongated physiology. Leptospira is generally considered to be an environmentally-associated pathogen where human infection is strongly linked to flooding events or outside contamination of surface waters from animal urine. Outbreaks of leptospirosis generally do not occur in regions with proper sanitation practices and may be a reason for the lack of data in engineered systems. What published data is available on Leptospira in sanitation and related systems is summarized in the following subsections.

Overall, effective rodent control and urine management from various domesticated animals are important aspects in managing leptospires associated with sanitation systems.

Recommendations for a person in a developing region to manage Leptospira include:

Use chlorine tablets to treat drinking water. If no 1.

chlorine is available, heat the water past 50°C for at least one minute. Do not bathe in, or use recreationally, surface 2.

waters after flooding events or with known sources of fecal contamination. This is especially regarding individuals with open cuts or sores.

Compost manure/digestate if you plan on applying it 3.

to land or otherwise (see below section 3.1). Prevent livestock excrement from surfaces waters, 4.

outbreaks are traced to animal feces and urine in frequently used waters. Control pests like rats, mice, and other rodents by 5.

improving food storage and garbage disposal conditions.

Wastewater Treatment

On-site waterless sanitation

The reduction of Leptospira spp. being introduced into the environment, originating from either human or animal, would help in reducing the frequency of human contact with pathogenic serotypes. Over a third of the world does not have access to adequate sanitation facilities [START_REF] Who | Guidelines for the safe use of wastewater, excreta and greywater[END_REF], and open defecation is still a common practice in some of these regions. Because of the direct link between flooding events and outbreaks of Leptospirosis, there is further emphasis made on the physical containment of unabated excreta from entering the environment. During the rainy season, these uncontained wastes allow runoff into surface waters where they collect and allow for transmission to humans downstream. The implementation of basic sanitation infrastructures can vastly aid in the overall reduction and prevention of waterborne pathogenic Leptospira transmission. The application of basic types of toilets or pit latrines to create physical separation between waste streams and humans would be considered best practice for hygiene and sanitation management of leptospires. The pathogen content of urine is much less than in feces [START_REF] Höglund | Microbial risk assessment of source-separated urine used in agriculture[END_REF], suggesting the prioritization of solids partitioning of human waste if at all else.

Composting

Composting is considered an effective, passive, waterless approach to reducing the overall biological activity and concentration of pathogenic bacteria in highly concentrated wastes and has been found to be effective in reducing pathogenic Leptospira. The main factors influencing the inactivation of Leptospira spp. in sludge and manure composting are the temperature and pH characteristics of the compost. [START_REF] Parker | Survival of a pathogenic Leptospira serovar in response to combined in vitro pH and temperature stresses[END_REF] simulated the pH and temperature profiles traditionally occurring during composting, evaluating their effect on the survivability of the pathogenic leptospires. Via logistic regression, they concluded that a temperature profile above 45°C for at least 4 hours has the potential to kill leptospires. Thermophilic temperatures >50°C for composting would then have the potential to kill leptospires in a few seconds, given that the entirety of the waste is subjected to comparable temperatures [START_REF] Chang | Studies on Leptospira icterohaemorrhagiae; survival in water and sewage; destruction in water by halogen compounds, synthetic detergents, and heat[END_REF][START_REF] Parker | Survival of a pathogenic Leptospira serovar in response to combined in vitro pH and temperature stresses[END_REF]. Leptospires have a slight favorability towards alkaline pH profiles [START_REF] Smith | The effect of pH on the survival of leptospires in water[END_REF], but in regards to the heterogeneity of compost matrices, the pH dependency of Leptospira survivability seems to dissolve [START_REF] Parker | Survival of a pathogenic Leptospira serovar in response to combined in vitro pH and temperature stresses[END_REF]. Composting would be considered a viable and effective solution for the management of leptospires in manure. [START_REF] Diesch | Survival of leptospires in cattle manure[END_REF] performed a study simulating an oxidation ditch being fed cattle manure from infected animals at a 1:10 scale laboratory model as well as field sampling. Leptospires were identified via dark-field microscopy in the aerated portion of the ditch for >60 days during summer temperature profiles as compared to 5 days in the anaerobic portion of the sludge. This suggests that available dissolved oxygen is critical to the survival of leptospires in comparable matrices. The study ended before a final contact time could be established for the complete removal of leptospires from the aerated portion.

Oxidation ditch

Waste stabilization ponds

Lined or unlined waste ponds are used worldwide to passively treat wastewater streams. Depending on the climate of the region, the effluents of these stabilization ponds can vary greatly, with a preference for warmer weather that allows for higher kinetic removal rates [START_REF] Hickey | Effluent characteristics of dairy shed oxidation ponds and their potential impacts on rivers[END_REF]. Although specific reductions in Leptospira concentrations in waste stabilization ponds have not been documented, it can be inferred that the level of dissolved oxygen in these ponds would dictate the efficacy of Leptospira reduction. Being that this a passive technique (no mechanical mixing or aeration), the dissolved oxygen content would be minimized in the lower portions of the stratified pond where a reduction of active leptospires would be anticipated.

Wetlands

Wetlands or "constructed wetlands" are artificial environments used to treat either municipal or industrial wastewaters. These systems are designed for the passive reduction of nutrient loadings on the environment by mechanisms already present in a natural ecosystem (nitrification, denitrification, phosphorus removal). These systems are not designed nor intended for pathogen removal and would not be considered a viable alternative. The persistence of leptospires in wet and aerated soils and sediments is well documented.

Aerated lagoons

No reports of Leptospira spp. reductions by aerated lagoons were identified.

3.1.6 Wastewater treatment facilities 3.1.6.1 Primary/preliminary treatment No reports of Leptospira spp. reductions by primary/preliminary treatment were identified.

3.1.6.2 Activated sludge Morphological spirochetes have been found in the activated sludge of treatment facilities in Amsterdam, but specific structural markers of the Leptospira genus were not found [START_REF] Deinema | Morphological characteristics of a spirochete present in activated sludge[END_REF]. Concentrated levels of spirochetes were seen in the interior of aggregated flocs and as motile cells on the floc surfaces. Being that Leptospira is an aerobic and chemoheterotrophic organism, it is inferred that the activated sludge portion of a treatment facility engenders their proliferation. The absence of oxygen, even in otherwise favorable growth conditions, will inhibit the survivability of leptospires in manure and sludge matrices [START_REF] Diesch | Survival of leptospires in cattle manure[END_REF]. It can then be inferred that anaerobic digestion of sludges would be a favorable environment for the eradication of infective leptospires. A study done by [START_REF] Mcgarry | Compost, fertilizer, and biogas production from human and farm wastes in the People's Republic of China[END_REF] investigated the effect of biogases on the survivability of Leptospira icterohaemorrhagica, finding that the spirochetes died within 31 hours after introduction of biogas as compared to 300 hours in the control.

Disinfection as a Tertiary (or Post Primary) Treatment and Drinking Water

Chlorine disinfection

As a tertiary treatment method for wastewater, chlorine appears to be the most relied upon treatment for the eradication of pathogens, including Leptospira, and is the recommended approach by the EPA (USEPA, 1999). Depending on the chlorine demand of the secondary effluent matrix, the recommended dosage range for tertiary treatment is 5-30 mg/L. These values are corroborated by a 1948 study where all leptospires were killed within one minute at a neutral pH and chlorine residual of 3.5 mg/L, or within three minutes at a residual of 1 mg/L [START_REF] Chang | Studies on Leptospira icterohaemorrhagiae; survival in water and sewage; destruction in water by halogen compounds, synthetic detergents, and heat[END_REF]. Unfortunately, specific Ct (concentration x time) values could not be identified.

UV disinfection and natural processes

Leptospires are also considered highly susceptible to UV light and Leptospira cultures were killed by direct exposure to sunlight for 1-2 hours in distilled water [START_REF] Faine | Leptospira and Leptospirosis[END_REF]. In a study, exposure to UV light (10 J/m2) resulted in inactivation of most pathogenic Leptospira strains in contrast to non-pathogenic leptospires which were more resistant to the same UV radiation dose; this study also showed that Leptospira is more sensitive to UV radiation than other bacteria such as E. coli [START_REF] Stamm | Sensitivity of pathogenic and free-living Leptospira spp. to UV radiation and mitomycin C[END_REF].

  3.1.1.1 Pit Latrines, vault toilets, dry toilets

  3.1.6.3 Membrane bioreactors/trickling filtersNo reports of Leptospira spp. reductions by membrane bioreactors or trickling filters were identified.3.1.6.4 Anaerobic digestion and biogas

Table 1 . Incidence of diagnosed disease for virulent Leptospira Geographical Area of Study Gender Incidence per 100,000 Inhabitants a Year of Data Collection

 1

	World-wide	Both	1.03E+06 cases /year	1970 to 2008
	Africa	Female	8.6	1970 to 2008
	Africa	Male	25.5	1970 to 2008
	Americas	Female	6.6	1970 to 2008
	Americas	Male	23.3	1970 to 2008
	Asia and Oceania	Female	10.9	1970 to 2008
	Asia and Oceania	Male	37.5	1970 to 2008
	Europe	Female	2.3	1970 to 2008
	Europe	Male	7.8	1970 to 2008
	Source: Costa et al., 2015

a per 100,000 inhabitants unless otherwise noted but, no data available for prevalence or seasonality

Table 2 . Pathogenic Leptospira occurrence in sewage and wastewaters Area Year Sample Description Occurrence Percent Positive (Sample Numbers) Detection Method Reference

 2

	Brazil	2011 to
	(Salvador, Pau da	2012 (July to	Sewage Water	36% (121/335)	LipL32 qPCR on 40 mL water sample	Casanovas-Massana et al., 2018
	Lima)	January)
					Culture enrichment
	Colombia (Cordoba)	2009 to 2011	Waste water (from pig stable)	5.56% (1/18)	0.5 mL with and without filtration	Calderon et al., 2014 ;
					through 0.45µm filter
	UK (London)	1977	Sewage (cattle market and farms)	100% (2/2)	Culture of 20 mL serogroup Tarassovi identified	Coghlan and Kmety, 1987
					Culture	a 20ml reduced
					to 0.2ml
	UK (Oxford)	1980 to 1981	Sewage and sewage sludges	15.9% (30/189)	Via centrifugation/filtration	Jones et al., 1981
					tarrassovi serotype
					identified

Table 3 . Pathogenic Leptospira occurrence in water samples

 3

				Occurrence
	Area	Year	Sample Description	Percent Positive (Sample	Detection Method	Reference
				Numbers)
	Brazil (Pelotas)	NR a	Stagnant Water (abandoned swimming pool with dead possums and rats)	100% (1/1)	Culture (Few Drops) Forster et al., 2013
	Brazil (Rio de Janeiro, Petropolis)	2009 (May to July)	Community water supplies, small lakes, and surface water	0.1% (1/100)	Multiplex PCR on 125 pathogenic species mL lipL-32 and 16srRNA both genes amplified in	Vital-Brazil et al., 2010
	Brazil (Salvador, Pau da Lima)	2011 to January) 2012 (July to	Standing Water	34% (82/250)	lipL32 qPCR on 40 mL water sample	Casanovas-Massana et al., 2018
		2010 to
	Chile (Los Rios)	2012 (November	Puddles	19.3% (36/306)	lipL32-PCR on 50mL to 1.0L water	Muñoz-Zanzi et al., 2014
		to April)
		2010 to
	Chile (Los Rios)	2012 (November	Water in open containers	45.5% (10/22)	lipL32-PCR on 50mL to 1.0L water	Muñoz-Zanzi et al., 2014
		to April)
		2010 to
	Chile (Los Rios)	2012 (November	Water Canals/Rivers	3.9% (4/103)	lipL32-PCR on 50mL to 1.0L water	Muñoz-Zanzi et al., 2014
		to April)
		2010 to
	Chile (Los Rios)	2012 (October to	Puddles	27.3% (84/208)	PCR and sequencing on 50 mL to 1L water	Mason et al., 2016
		April)
		2010 to
	Chile (Los Rios)	2012 (October to	Water in open containers	14.4% (28/194)	PCR and sequencing on 50 mL to 1L water	Mason et al., 2016
		April)
		2010 to
	Chile (Los Rios)	2012 (October to	Water Canals/Rivers	9.7% (13/134)	PCR and sequencing on 50 mL to 1L water	Mason et al., 2016
		April)
					Culture enrichment
	Colombia (Cordoba)	2009 to 2011	Well Water	0% (0/18)	0.5 mL with and without filtration	Calderon et al., 2014
					through 0.45µm filter
					15 mL water samples
					collected on farms
	Colombia (Cordoba)	NA b	Untreated water samples	0% (0/13)	Centrifuged 14,000 rpm / 5 min. Pellet	Ensuncho-Hoyos et al., 2017
					washed by PBS. PCR
					and sequencing.

b

Isolated from blood cultures of moribund animals

Table 5 . Pathogenic Leptospira occurrence in drinking water samples

 5

				Occurrence
	Area	Year	Sample Description	Percent Positive	Detection Method	References
				(Sample Numbers)
		2010 to
	Chile (Los Rios)	2012 (November	a Animal drinking water	14.5% (12/83)	lipL32-PCR on 50mL to 1.0L water	Muñoz-Zanzi et al., 2014
		to April)
		2010 to
	Chile (Los Rios)	2012 (November	Human drinking sources	19.1% (9/47)	lipL32-PCR on 50mL to 1.0L water	Muñoz-Zanzi et al., 2014
		to April)
	Chile (Los Rios)	2010 to 2012 (October to April)	Animal drinking water a	16.5% (14/85)	PCR and mL to 1L water sequencing on 50	Mason et al., 2016
	Chile (Los Rios)	2010 to 2012 (October to April)	Human drinking sources	14.7% (14/95)	PCR and mL to 1L water sequencing on 50	Mason et al., 2016
	China (Nanjing, Bejhekou drinking plant) water	NA b	Drinking Water	0.45% of total metagenomic reads	Metagenomics (454 chlorination pyrosequencing) in tap water after L. interrogans still	Huang et al., 2014
					Culture enrichment
	Colombia (Cordoba)	2009 to 2011	Animal drinking water a	5.56% (1/18)	0.5 mL with and without filtration through 0.45µm	Calderon et al., 2014
					filter

a Non-potable drinking water for animals; b NA: Not Available

Table 6 . Persistence of Leptospira in different matrices

 6

	Area (Year)	Starting Concentrations (Species)	Matrix/ Temperature (°C) Conditions	Maximum Survival in Days	Reference
	New York (1918)	Unknown (L. interrogans serogroup Icterohaemorrhagiae)	East river water	<1	Noguchi, 1918
	New York (1918)	Unknown (L. interrogans serogroup Icterohaemorrhagiae)	sewage water	<1	Noguchi, 1918
	New York (1918)	Unknown (L. interrogans serogroup Icterohaemorrhagiae)	stagnant water	<1	Noguchi, 1918
	New York (1918)	Unknown (L. interrogans serogroup Icterohaemorrhagiae)	horse stool emulsion	<1	Noguchi, 1918
	New York (1918)	Unknown (L. interrogans serogroup Icterohaemorrhagiae)	sewer filtrate	<1	Noguchi, 1918
	New York (1918)	(Pathogenic Leptospira strain Flanders)	non sterile distilled water with few large motile bacilli	3	Noguchi, 1918
	New York (1918)	(Pathogenic Leptospira strain Flanders)	drinking water	7	Noguchi, 1918
	North Queensland, Brisbane, (1950s) Australia	20 mL of a 7 day culture (strain Australis A) into soil Pathogenic Leptospira	Soil	43 days in soil 3 days in water	Smith and Self, 1955
	North Queensland, Brisbane, (1950s) Australia	20 mL of a 7 day culture into soil (strain Australis A) Pathogenic Leptospira	Rain water from soil	15 days in soil 4 days in water	Smith and Self, 1955