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. 

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

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

o   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.

o   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.

o   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%).

o   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.

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

o   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.

o   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.

Leptospirosis

Leptospires enter the host via small abrasions, cuts of the skin, conjunctiva, mucous membrane and genital tract. The bacteria enter blood stream or remain in the kidney tubules and be shed in the urine for a period of a few weeks to several months and even longer. 

Once the number of Leptospires in the blood and tissues reaches a critical level, lesions and other symptoms appear, due to the action of  leptospiral toxin(s) or toxic cellular components .

Endotoxin activity due to Leptospiral LPS (leptospiral lipopolysaccharide) has been reported as seen in other Gram-negative bacteria. Hemolysins such as phospholipases act on erythrocytes and other cell membranes containing phospholipids, leading to cytolysis.

 Pathogenesis

•      In natural reservoir hosts – infection asymptomatic. 

•      If infection is transmitted to other animals, cause clinical disease

•    Human infection- Leptospires enter the body through cuts, abrasions on the skin or through intact mucosa of mouth, nose or conjunctiva, when water is contaminated by urine of carrier animals

•      Incubation period 2-26 days (10 days)

         Symptoms

•  Mild undifferentiated pyrexia (fever) to Severe or fatal illness with hepatorenal damage (Weil’s disease)

•   In severe cases, the onset is acute, with rigor (stiffness), vomiting, headache and irritation of the eyes- retro-orbital pain (around orbit of the eyes), conjunctival redness etc.

•     The fever is irregular and subsides in about 10 days

•      Jaundice by the second or third day in 10-20 percent cases

•      Purpuric hemorrhages (small spots of blood appear on skin) - on the skin and mucosa

•      Albuminuria is common

Leptospirosis is of two clinical types- icteric (with fever) and non-icteric

•     Meningitis is common and in some, abdominal symptoms predominate. Hepatorenal failure, hemorrhage of skin and mucous membrane, jaundice, and myocarditis also seen.

•      Clinical diagnosis is impossible in majority of cases –misdiagnosis common

•      High vigilance and laboratory assistance important not to miss out leptospirosis identification

Serious cases of human leptospirosis caused by the serotype L. icterohemorrhagiae. Aseptic meningitis common in L. canicola infection. Abdominal symptoms in L. grippotyphosa infection.

But, clinical syndromes are not serotype specific- Any type of illness may be produced by any serotype.

Epidemiology

•      Leptospirosis- most widespread of zoonoses

•      Present in all continents, except Antartica

•      Pathogenic leptospires survive for long periods in (the convoluted tubules of) the kidneys in natural hosts, multiply and are shed in urine

•      Animal carriers excrete upto 100 million leptospires per ml of urine

•      Infected urine contaminates mud/water that is neutral or slightly alkaline-leptospires survive for weeks

•      People come into contact with such water-leptospires enter the body through abraded skin or mucosa and initiate infection

•      Certain occupational groups-agricultural workers in rice/cane fields, miners, sewage cleaners exposed to infection-more common in them

•      Leptospires can be shed through milk of lactating animals-die rapidly in milk

•      Human infection through milk unknown

•      Not shed in saliva-animal bites not infectious;   Arthropods do not transmit the infection

•      Animals as carriers- rats-ubiquitous, carry the most pathogenic serotype icterohemorrhagiae

•      Field mice carry grippotyphosa, pigs carry pomona, dogs carry canicola serotypes

•      Generally nonpathogenic in the reservoir animal

•      Of veterinary importance since infection in cattle/pigs cause economic loss

•      Infection among animals- transmitted by contaminated water/fodder

•      Human beings are an aberrant/end host- no transmission to other humans

•      Was a rural disease of agricultural workers-  Now, an urban problem in developing countries- overcrowding, insanitation, increasing rat population and habit of walking barefoot

 

Laboratory diagnosis 

Leptospires are seen in the blood during the acute phase of the disease but can seldom be demonstrated after 8-10 days. They persist in the internal organs, and most abundantly in the kidneys, so may be demonstrated in the urine in the later stages of the disease.

v  Microscopic Demonstration in blood/urine

v  Isolation in culture

v  Inoculation in Guinea pigs

v  Serological tests

 

Examination of Blood

•      Helpful in early stages of the disease (before antibiotics are given)

•      Leptospires disappear from blood after the first week

1) Examination of blood under dark field microscope/by immunofluorescence; but of little practical value

 

2)Three or four drops of blood are inoculated into EMJK or similar medium- incubated at 37⁰C for two days and left at room temperature in the dark for two weeks

•      Samples from the cultures are examined every third day for the presence of Leptospires under dark ground illumination

•      Primary isolation may be delayed and may take many weeks to months

•      Chances of isolation are increased by culturing blood daily at the early stage of the disease

•      Leptospires may be isolated from CSF also

3)      The blood from the patient is inoculated intraperitoneally into young guinea pigs

With virulent serotypes- icterohemorrhagiae, the animals develop fever, die within 8-12 days with jaundice and hemorrhage into the lungs and serous cavities

With other serotypes- canicola or pomona, animal may not become ill-Leptospires should be demonstrated in the peritoneal fluid, by blood culture or by serology

From the third day after inoculation, the peritoneal fluid is examined daily under dark ground illumination

When Leptospires are detected the blood withdrawn by cardiac puncture is inoculated into culture media

 

 Urine Examination

• Leptospires appear in the urine in the second week of the disease and intermittently thereafter for 4-6 weeks
• Urine should be examined immediately - Leptospires readily undergo lysis in acid urine

1)      Centrifuged deposit of urine is examined under dark field microscope

2)      Direct culture of urine seldom successful due to contamination but isolation is possible by inoculation into guinea pigs

 

Serological tests

• Antibodies appear in serum towards the end of the first week of the disease, increase till the fourth week, then reduces.
• Antibodies/Agglutinins are demonstrable years after the infection
• Serological tests can be- broadly reactive genus specific screening tests and the serotype specific tests

v  Genus specific/ Broadly reactive tests -identify Leptospiral infection without identifying the exact serotype

•      The antigens for these tests are prepared from the nonpathogenic L. biflexa Patoc 1 strain

•     Sensitized erythrocyte lysis (SEL), complement fixation, agglutination and indirect immune florescence, ELISA

•      ELISA to detect IgM and IgG separately, to determine the stage of infection can be done

•      Simple and rapid dip stick assays for detecting Leptospira specific IgM antibody in human sera available

v  Serotype specific tests

•      Identify the infecting serotype by macroscopic and microscopic agglutination tests

•      Macroscopic agglutination tests done with inactive formalinized suspensions of Leptospiral serotypes with serial dilutions of the test serum

•   Microscopic agglutination tests (MAT) use live cultures of different serotypes and agglutination is observed under the low power dark field microscope- more specific- done in reference laboratories

 

Prophylaxis

•      Leptospirosis results from contact of skin or mucosa with contaminated water

Ø  Rodent control

Ø  Disinfection of water

Ø  Wearing of protective clothing

•      Vaccination of high risk groups such as agricultural workers encouraged.  Immunity is serotype specific

Ø  Vaccination of dogs, cattles, pigs

 

Therapy

•      Sensitive to penicillin and tetracyclines

•      Treatment should be started early in the course of the disease

•      Penicillin given as IV : 1-2 million units 6 hourly for 7 days, for serious cases

•       A mild Jarish Herxheimar reaction seen in some

•      Doxycycline 200 mg orally given once in a week effective for prophylaxis

 

Jarish Herxheimar reaction - common detoxification response of the body to the increased toxins released during a treatment for pathogens

•      the amount of toxins released into the circulation is more than what our body can safely handle quickly -      lot of stress on internal organs, especially the colon, liver and kidneys that are involved in filtering toxins in the body