Leptospira - Morphology, Cultural characters

Leptospira- Order Spirochaetales, family Leptospiraceae, genus Leptospira.

Leptospirosis is infection with the Spirochaete Leptospira. It is an acute zoonotic infection of worldwide significance. Leptospirosis is seen in both humans and animals.  The primary reservoir is rodents such as rats, mice, wild rodents and once infected, they shed the organisms for life. 

Livestock farming is the major occupational risk factor for human leptospirosis since cattle, dogs, swine etc., can also be reservoirs.

                                   

Infected animals excrete Leptospira both in active infection and asymptomatic stage. The Leptospira survive and remain viable for several weeks in stagnant water. It is common in temperate or tropical climates - rare in North America. Transmission to humans occurs through penetration of the organism into the blood stream via cuts, skin abrasions or mucus membranes

                                                    

Leptospirosis is also known as hemorrhagic jaundice, infectious jaundice, mud fever, spirochetal jaundice, swamp fever, swineherd's disease, caver's flu, sewerman's flu, Canicola fever (canine leptospirosis-dogs) etc

First human leptospiral disease was described by Adolf Weil in 1886, as an "acute infectious disease with enlargement of spleen, jaundice and nephritis. Leptospira was first observed by Stimson in 1907 from a post mortem renal tissue slice. Stimson named it Leptospira interrogans owing to its shape resembling interrogation (question) mark.

                          

Human infection also known as Weil's disease, is caused mainly by Leptospira icterohaemorrhagiae, which was isolated in 1915 by Inada. Consequently many leptospires have been isolated.

Spirochetes are divided into two families, Spirochaetaceae and Leptospiraceae. 

• Spirochaetaceae –include Treponemes, Serpulina and Borrelia 
• Leptospiraceae include Leptospira.

Leptospira is further classified into several species and subspecies, called serogroups and serovars, based on the surface (lipopolysaccharide -LPS) antigens. 

Genus Leptospira is divided into two species 

• L. interrogans includes pathogenic strains 
• L. biflexa includes saprophyte strains -from the environment. 

These two species are divided into serovars  as defined by agglutination techniques. There are approximately 60 serovars for L. biflexa, more than 200 for L. interrogans.

L. interrogans has more than 22 serogroups common examples being L. icterohaemorrhagiae, L. Canicola, L. australis, L. hebdomadis, L. andamana, L. pyrogenes etc

Morphology

Leptospires are corkscrew-shaped bacteria, delicate flexible helical cells, about 0.1 µm in diameter by 6–20 µm in length, which differ from other spirochaetes by the presence of end hooks. They have numerous coils with ends hooked like umbrella handles. Actively motile, with single polar flagellum.

Stains poorly with aniline dyes.

Observed using Giemsa stains, fluorescent antibody techniques or by silver impregnation methods.

Best observed using dark field microscopy or phase contrast for observation under living conditions and otherwise by, electron microscopy due to narrow size.

Growth characters/Cultural characters

            Leptospires are aerobes and microaerophiles with an optimum growth temperature of 25–30°C and pH 7.2-7.5.

They grow in  media enriched with vitamins B1 and B12, long-chain fatty acids, and ammonium salts. Long-chain fatty acids are utilized as the sole carbon source and are metabolized by β-oxidation.

Media enriched with rabbit serum or several liquid and semisolid media such as Korthof’s, Stuart’s and Fletcher’s media, etc. can be used. They grow in laboratory media within 12-16 hrs. and in inoculated animals within 4-8 hrs.

Growth of leptospires may be slow on primary isolation, and cultures have to be retained for about 13 weeks before being discarded. Agar may be added at low concentrations (0.1– 0.2%).

Semisynthetic media such as EMJH : Ellinghausen-McCullough-Johnson-Harris medium which contains fatty acids and bovine serum albumin is commonly used. In such semisolid media, growth is seen beneath the surface of the medium, which becomes more turbid with incubation time. This growth is known as Dinger’s ring or disk.

 

Leptospires can also be grown on chorioallantoic membrane (CAM) of chick embryos –growth observed in 4-5 days.

            Bacterial contamination in cultures prevented by using antibiotics. Inoculation of the sample intraperitoneally in gunea pigs and culturing the heart blood collected after ten minutes is a good method to obtain contaminant free cultures. Leptospires invade blood stream more readily than other bacteria.

Leptospiral cultures are maintained by repeated subculture or by storage in semisolid agar containing hemoglobin. Long-term storage in liquid nitrogen also yields good results and is the preferred method of storage for maintaining virulence.