Leptospirae: An overview
▶The History of Discovery
The first observations of icteric leptospirosis with renal failure were registrated in 1886 by A. Weil in Germany, though the similar disorders were described still in ancient ages (e.g., the disease of rice harvesters in China or autumn fever in Japan).
In 1907 A. Simpson discovered the presence of hook-ended spirochetes in the kidney specimen of patient, who supposed to die from yellow fever. He called them Spirochaeta interrogans, as they resembled question mark.
Only in 1915 these agents were re-discovered by R. Inada and R. Ido in Japan, who isolated bacteria from the blood of Japanese miners with infectious jaundice, and almost at the same time by two independent groups of German researchers (P. Uhlenhuth and W. Fromme; E. Hubener and H. Reiter) after examination of German soldiers in northeast France that suffered from so-called “French disease” during World War I.
▶Modern Classification of Leptospirae
Leptospirae pertain to the order Spirochaetales, family Leptospiraceae, and genus Leptospira.
Recently classification of leptospirae has been greatly changed within the borders of the same genus. Before early 1990s only two leptospira species have been distinguished, L. interrogans that contained all pathogenic serovars, and L. biflexa, encompassed all environmental saprophytic strains. Further division was grounded on microbial serologic properties. L. interrogans comprised more than 200 serovars, while L. biflexa included about 60 serovars. Bacterial serovars were consolidated into various serogroups.
With the progress of bacterial genetic typing, genotypic classification of leptospirae substituted the serological division, and numerous genomospecies accumulated the serovars of initial species, L. interrogans and L. biflexa.
More than 20 genetic species of leptospirae are distinguished now. In this classification pathogenic and saprophytic serovars can be placed into the same genomospecies. Moreover, former species L. interrogans sensu lato and L. biflexa sensu lato don’t coincide with the same name genomospecies. Therefore, previous phenotypic classification lacked correspondence with modern genetic typing of leptospirae, albeit serological division remains convenient for practical use and retains its value for seroepidemiological studies.
Main genomospecies and serogroups of leptospirae are present in table.
▶Structure and Properties of Leptospirae
Leptospirae have very thin cell structure, usually 0.1 by 20 μm in size. These spiral bacteria make multiple turns around the axial filaments, thus forming small primary coils. Being supercoiled, leptospirae produce secondary twists with distinctive hooks at their ends, shaping interrogative mark, or letters C and S under microscopy. The organisms bear two axial filaments (periplasmic flagella) attached at opposite ends to basal bodies within periplasmic space. Flagella ensure striking motility of microbial cells.
The bacterial genome was shown to be composed of two parts: large 4,400 kb chromosome and small 350 kb chromosome. No other plasmids were described.
Leptospirae don’t contain spores and capsules. Microbial body is encased within the outer membrane. Bacterial lipopolysaccharide is similar with other spirochetes, but shows lower endotoxin activity.
Leptospirae poorly accept aniline dyes due to their compact structure and large lipid contents. These bacteria are gram-negative; also they stain pinkish with Romanowsky-Giemsa method. The best technique of bacterial visualization is dark field microscopy. Silver impregnation or Burri stain with Indian ink background may be applied as well.
Leptospirae are cumbersome for culture. Optimal growth temperature for cultivation is 28-30°C. Bacteria propagate slowly in liquid and semisolid media, e.g. Vervoort-Wolff, Fletcher, Noguchi and others. The media contain serum or albumin, vitamins, long-chain fatty acids, and ammonium salts. Several synthetic protein-free media were elaborated, e.g. complex oleic acid-albumin medium is used. Primary growth appears in several weeks of cultivation, subcultures grow within 1-2 weeks.
- Biochemical properties
Leptospirae are obligate aerobic bacteria. They produce catalase and oxidase.
As many other spirochetes, leptospirae have slow metabolism, which is not completely elucidated. Bacteria use exclusively long-chain fatty acids as the only source of carbon. They can’t utilize peptides and carbohydrates as energy supplies. Ammonium salts are used as the source of nitrogen.
- Antigenic structure
Antigenic composition of leptospirae is complex and renders great cross-reactivity between various serogroups and serovars.
The outer membrane of bacteria contains LPS antigen and various lipoproteins, i.e. outer membrane proteins (OMPs) that show antigenic activity.
Microbial LPS confers serovar specificity.
- Virulence factors
Leptospirae carry various adhesins promoting microbial attachment to host cells and tissues, e.g. renal epithelial cells.
Bacteria possess low but distinct endotoxic activity of LPS. Microbial LPS also stimulates platelet aggregation during infection.
Leptospirae are capable of producing several hemolysins, some of them show sphingomyelinase or phospholypase activity. Particular strains elaborate limited number of cytotoxins of protein or glycolipoprotein nature.
Bacteria express some antiphagocytic substances, and fibronectin-binding protein that hinders microbial opsonization.
Leptospirae show resistance to low temperatures, alkaline pH and stay viable in water reservoirs for many months. They are very sensitive to drying and acids. Heating at 56°C for 30 minutes inevitably kills bacteria. Leptospirae are easily lysed in bile-containing media. They are sensitive to standard disinfectants.
▶Pathogenesis and Clinical Findings in Leptospirosis
Leptospirosis is a typical zoonotic disease. It is regarded as one of the most widespread zoonosis.
The disease is transmitted predominantly via direct contact of susceptible host with the urine of infected animal. The incidence of disease is greatly increased in tropical countries with moist climate with maximal incidence in summer or rainy seasons.
The main sources of infection are rodents (e.g., rats), which may contract infection to domestic animals (e.g., cattle), dogs, and other mammals. They can appear to be additional infection source for humans.
Leptospirosis is an occupational disease. The increased risk of illness is reported in farmers, fish workers, sewer workers, veterinarians, miners, soldiers and others.
The causative agent usually enters the body through skin lesions or cuts. Also it can penetrate conjunctiva. Long exposure to infected water may provide infection through intact skin. Waterborne and foodborne transmission is possible via contaminated water and foodstuffs. Likewise, inhalation of water aerosol may produce infection.
Incubation period lasts for about 1-2 weeks.
A great number of leptospirosis cases are subclinical or mild. Nevertheless, about 5-10% of patients develop life-threatening icteric leptospirosis with sudden onset, fever, severe headache, transient rashes, myalgia, and abdominal pain. The fever may be biphasic with relapse in 3-4 days. Bacteremia emerges in the first days of illness.
The disease is characterized by profound injury and dysfunction of most inner organs. It ensues from generalized infectious vasculitis caused by leptospirae that is followed by endothelial damage and tissue inflammation.
Manifested infection results in infectious hepatitis with jaundice and high serum bilirubin level that maintains for a long time. Up to 40% of affected persons produce acute renal failure due to kidney tubular damage. Necrotizing pancreatitis, lung and cardiac involvement followed by pneumonia and myocarditis are also characteristic for disease. Aseptic meningitis appears in quarter of patients.
If not terminated by efficient treatment, the disease comes into second immune phase. It is followed by bacterial disappearance from the bloodstream with the rise of specific IgM antibodies. Autoimmune mechanisms contribute to disease progression. Various autoantibodies, including anticardiolipin and antineutrophil cytoplasmic antibodies appear in the disease course. Accumulating immune complexes promote complement activation and cell-mediated cytolysis that enhances tissue damage.
Disease lethality in case of icteric leptospirosis varies within 5-15%.
High levels of specific antibodies ultimately cause bacterial elimination that lead to patient recovery. Nevertheless, shedding of viable bacteria with urine is possible long after clinical convalescence.
The disease confers long lasting stable immunity, which is largely maintained by specific antibodies. In most cases the protection is serovar-specific.
▶Laboratory Diagnosis of Leptospirosis
Specimens are collected from patient’s blood, urine, tissue aspirates, or cerebrospinal fluid, which are used for microscopy and culture. Serum is taken for serological tests.
Dark field microscopy can reveal about 104 leptospirae/ml. Sample centrifugation can increase the sensitivity of test. Immunofluorescence technique or light microscopy with Giemsa stain are also used to visualize leptospirae in blood or urine. Microscopy of blood can be positive only at the first few days of the disease in bacteremia stage.
Serologic determination of leptospiral antigens in clinical specimens by ELISA provides higher sensitivity and accuracy comparing with microscopic methods.
Bacterial culture is difficult in routine practice. Patient’s blood should be taken only within the first days of the disease in bacteremia stage. Urine is tested from the second week of the disease onset.
Samples are inoculated into special nutrient media. Cultures are tested weekly by dark field microscopy for up to 13 weeks. Identification of bacteria is improved by serological methods or molecular tests (PCR). Faster detection of leptospiral growth is possible by radiometric methods.
To accelerate microbial isolation intraperitoneal inoculation of hamsters or guinea pigs with patient’s material is elaborated. Leptospirae can be detected in peritoneal cavity of infected animals at the end of the first week after inoculation.
Serological tests prevail in laboratory diagnosis of leptospirosis. Various methods are applied to clinical practice.
In case of microscopic agglutination test (MAT) patient’s serum containing specific antibodies is incubated with antigenic mixture of various live leptospiral serovars. After incubation the reaction is evaluated mainly by dark field microscopy. The titer or end point of the reaction is the highest dilution of serum, where 50% agglutination of leptospirae occurs. Antibody titers of 1/200-1/400 are regarded as the positive result. Acute infection elicits much more high titers of specific antibodies (even greater than 1/25,000).
Complement fixation test, indirect hemagglutination and ELISA are also used for serological diagnosis.
Molecular methods, including DNA and RNA hybridization and PCR show highest sensitivity but can’t determine the serovar of isolated bacteria.
▶Specific Prophylaxis and Treatment of Leptospirosis
Early vaccines for specific prophylaxis of leptospirosis contained the mixture of inactivated leptospirae cultured in serum media. After injection they provoked various side effects. Modern vaccines are obtained from serum-free media and include a number of the most clinically significant serovars (L. icterohaemorrhagiae, L. canicola, L. grippotyphosa, and others). These biological products can be used for vaccination of domestic animals as well as for protection of humans from groups of risk.
Antibiotic treatment of the disease should be started as soon as possible. Beta-lactams (amoxycillin, cephalosporins) and doxycycline are regarded as the most effective drugs for leptospirosis treatment. Patients with acute renal failure require urgent hemodialysis.