Leptospiria and Leptospirosis

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Alejandro de la Pena-Moctezuma (Universidad Nacional Autonoma de Mexico )Gabriel Trueba (Universidad San Francisco De Quito, Institute of Microbiology)


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.