Spirochaetes

        Elongated, motile, flexible bacteria twisted spirally along the long axis. A charecteristic feature is the presence of endoflagella-polar flagella, situated between outer membrane and cell wall. They are Gram negative of varying sizes. Many are free living saprophytes, while some are obligate parasites. They may be aerobic, anaerobic, facultative. Reproduction by transverse fission. Human pathogens are: Treponema, Borrelia, Leptospira. The spiral shape and serpentine motility of the spirochaetes depend upon the integrity of these endoflagella. Motility is of 3 types : (i) flexion & extension (ii) cork-screw like rotary movement (iii) translatory motion.

Treponema pallidum – Some of them are pathogenic for man, while others occur as commensals in mouth and genitalia. It is a thin, delicate spirochete with tapering ends. It is about 6-14 μm. Its width is 0.13 μm in dried state, 0.2 μm in the wet living state. It has about ten regular spirals. It is actively motile, exhibiting flexion & extension, translatory and corkscrew-like motility. It is observed under dark background in microscope. It often displays a characteristic tendency to bend at right angles near its midpoint.

It stains with Giemsa giving rose red. The cytoplasm is surrounded by a trilaminar cytoplasmic membrane, enclosed by a cell wall containing peptidoglycan. External to this lipid rich outer membrane. 3-4 endoflagella between cell wall and outermembrane. They do not grow in artificial culture media. Limited growth in tissue culture cells. It can be readily inactivated by drying, by 41-42°C heat. It is also inactivated by O₂, distilled water, soap, antiseptics, antibiotics. 

Cultivation – Pathogenic treponemes cannot be cultivated in artificial media and are maintained by subculture in susceptible animals. Nichol’s strain has been maintained, in rabbit testis, by serial testicular passages since 1913. The mean generation time for growth in the rabbit testis is 30 hours. It can be maintained, in vitro, for 1-6 days using maintenance medium that contains rabbit serum, reducing agents, vitamins, cofactors, amino acids and salts. Non pathogenic strains are cultivable and they can be grown at strict anaerobic conditions.

Susceptibility – T. pallidum is readily inactivated by heating at 41.5⁰C for one hour. When infected blood is stored at 5⁰C in citrate anticoagulant, infectivity is lost in 5 days or less. Therefore, transfusion syphilis can be prevented by storing blood for at least 5 days in the refrigerator by transfusion. Frozen in a medium containing 15% glycerol at –70⁰C, they remain viable for years and provide a reliable source of antigens for immunological tests, but freeze drying kills the organism.

Antigenic structure – On the basis of three types of antibodies produced on the treponemal infection, there are 3 different antigens.

ü  Group-specific antigen – It is a protein present in pathogenic Nicol’s strain and nonpathogenic Reiter’s strain.

ü  Species- specific antigen – It is polysaccharide in nature.

ü  Cardiolipin – A non specific antigen. A non specific antibody, reagin, appears in the blood of syphilitic patients that reacts with a lipid hapten extracted from the beef heart, this hapten is cardiolipin, and is chemically a diphosphatidyl glycerol.

Pathogenicity –T. pallidum is the causative agent of syphilis. It is a strict parasite and its life outside the animal body is short. The disease is transmitted by sexual contact, passed through body fluids such as saliva. So it is a hazard for dentists, dental hygienists. It is not transmitted in food, water, or air, by arthropod vectors. Humans are the only reservoir. Donated blood need not be screened for syphilis because any spirochaetes present are killed when the blood is refrigerated. In veneral syphilis, the treponemes penetrate mucosal surfaces or abraded skin and multiply at the site of entry and after the incubation period  the clinical disease sets on. The progression of syphilis has 6 stages:

1. Incubation Stage: Over a period of 2-6 weeks after entering the body, the organisms multiply and spread throughout the body

2. Primary Stage: An inflammatory response at the original entry site causes formation of a chancre, a hard, painless non discharging lesion about 1/2 inch in diameter. One or more primary chancre develop on lips or hands, genitals. A large number of treponemes are present in the primary lesion. In females, chancres on cervix or other internal location, sometimes escape detection. Even though it goes away in 4-6 weeks, without leaving any scarring, the disease has entered the next stage.          

3. Primary latent period: All external signs of the disease are disappear, but blood tests for syphilis are positive.

4. Secondary stage: Symptoms appear, disappear and reappear over a period of about 5 years, during which the patient is contagious. The symptoms include a copper-colored rash, particularly on the palms of the hands and the soles of the feet, and various rashes and skin erruptions. Painful, whitish mucus patches swarming with spirochaetes appear on the tongue, cheeks, gums. Lesions heals, as the disease has entered the next stage.

5. Secondary latent stage: All symptoms disappear and blood tests can be negative. This stage can persist for life or for a highly variable period, or it may never occur. In some, the disease does not progress beyond this stage, but in some it progresses to the next stage

6. Tertiary stage: Permanent damage occurs throughout various systems of the body. In this stage it mimics symptoms of many other diseases. Most involve the cardiovascular and nervous systems, thereby blood vessels and heart valves are damaged by calcium deposits in heart-valves.        

Neurological damage or Neurosyphilis, include thickening of meninges, inability to walk (Ataxia), paralysis, insanity. These symptoms are due to the formation of granulomatous inflammations or gummas. Internal gummas, destroy neural tissue, external gummas destroy skin tissue. Mental illness following neural damage.                                                                                               Congenital Syphilis occurs when treponemes cross the placenta from mother to baby. At birth or shortly thereafter, the infant may show signs such as notched incisors (Hutchinson's teeth), a preforated palate, saber shins(in which the shins bone or tibia projects sharply on the front of the leg), an aged looking face with saddle nose(a flat, saddle shaped nose) and nasal discharge.                   

Laboratory Diagnosis - It consists of demonstration of the spirochaetes under the microscope and of antibodies in serum or CSF.                                                

A. Microscopy: It is applicable in primary and secondary stages and in congenital syphilis with superficial lesions. Specimans should be collected with care as the lesions are highly infectious. The lesion is cleaned with a gauze soaked in warm saline and the margins gently scraped. Gentle pressure applied to the base of the lesion and the serum that exudes is collected preventing mixture of the blood. Wet films are prepared with the exudate and after applying thin coverslips, examined under dark-ground microscope. T. pallidum identified by its slender spiral structure and slow movement. Dark-ground examination can be negative, but it doesn't mean there is no syphilis. A treponemal concentration of 104 per ml is necessary in exudates, for the test to be positive. The direct fluorescent antibody test for T. pallidum (DFA-TP) is a better method for microscopic diagnosis. Smears of the exudate are fixed with acetone, and DFA-TP test done using fluorescent tagged T. pallidum antiserum.                     

B. Serological Tests: Mainly 3 serological tests.                                          

1. Reagin antibody tests: These tests use the lipoidal or cardiolipin antigens and are known as standard tests for syphilis or STS. The antibody reacting with cardiolipin is known as reagin. This is done by VDRL test(Veneral Disease Research Laboratory),which gives quantitative results. In this, inactivated serum is mixed with cardiolipin antigen on a special slide and rotated for 4 minutes. Cardiolipin remains as uniform crystals in normal serum but forms visible clumps on combining with reagin antibody. By testing serial dilutions, the antibody titre can be determined. Modification of VDRL test developed, Rapid Plasma Reagin(RPR) in which VDRL antigen containing fine carbon particles, which make the result more clear cut and evident to the naked eye. It is done with unheated serum or plasma. But since cardiolipin is present in T. pallidum and in mammalian tissues, reagin antibodies may be induced by treponemal as well as host tissue antigens and therefore Biological False Positive reactions or BFP reactions constitute the major disadvantage of STS. But it can be known by checking the type of immunoglobulin present in the sera, BFP antibody in syphilis is mainly IgG. BFP reactions are due to acute infections and chronic infections such as leprosy, malaria, hepatitis etc.

2.Group Specific Treponemal tests: This test is for antibodies reacting with group-specific treponemal antigen. It is known as Reiter protein complement fixation (RPCF) test, uses LPS-protein complex antigen derived from the bacteria. Here an adapted strain of treponeme is used, BFP reactions are low

3. Specific T. pallidum tests:  This test use virulent Nichol's strain of T. pallidum maintained by serial inoculation in rabbit testes. In this FTA-ABS and TPHA are used. The Fluorescent Treponemal Antibody (FTA) test is an indirect immunofluorescence test in which antigen smears prepared on slides with Nichol's strain. The modified form of this test is FTA-Absorption or FTA-ABS, in which the test serum is preabsorbed with Reiter treponemes (cultivable,non-pathogenic strains), FTA-ABS is specific and is accepted as a standard reference test. The T. pallidum Hemagglutination Assay(TPHA) uses, tanned erythrocytes sensitised with a sonicated extract of T. pallidum antigen. The test sera for TPHA are absorbed with a diluent containing components of Reiter's Treponeme, rabbit testis and sheep erythrocytes. TPHA is also a standard confirmatory test.

Prophylaxis and treatment : Veneral syphilis is world wide in distribution, and has not been able to eliminate because of changing customs habits and values in the society. As transmission is by direct contact, it can be minimized by avoiding sexual contact with an infected individual. Or using physical barriers, antiseptics, antibiotics minimize the risk. No vaccine is available. Penicillin is uniformly effective, but is necessary to give an adequate dose and maintain the drug level for a sufficiently long period. Erythromycin, tetracycline can be used in penicillin allergic patients. Recovery does not confer immunity in all.                                                                              

Borrelia - They are large, motile, refractile spirochaetes with irregular, wide open coils. They are usually 10-30 µm long and 0.3-0.7 µm wide. They are Gram negative. Several species occur as commensals on the buccal and genital mucosa. Two important forms are:  B. burgdorferi, B. recurrentis. These are transmitted to vertebrate hosts by hematophagous arthropods.                   B. burgdorferi : The causative agent of Lyme disease, identified in 1982. It was first observed in Lyme, USA, hence the name. It is an arthropod vector-borne infection, is microaerophilic. It is carried by Ixodes scapularis, the blacklegged tick, which feeds on deer and small mammals such as mice. The borrelia grows mainly in the midgut of the tick. Infection occurs by regurgitation of the gut contents during biting. Dogs, horses, cows and humans are infected.                                

Pathogenicity - Most patients develop flu-like symptoms after the bite. After an incubation period of 3-30 days, the first stage of "localised infection" appears as an expanding annular skin lesion/bull's eye rash at the site of the bite. A few weeks later, the second stage,"disseminated infection" with fever, headache, arthritis, myocarditis etc. occurs. Also loss of insulating myelin from nerve cells cause symptoms resembling Alzheimer's disease and multiple sclerosis. The third stage of "persistent infection" occurs after months or years later with chronic arthritis.

Laboratory Diagnosis - B. burgdorferi is a fastidious organism, so grown in a modified Kelley's medium, 2 weeks or more incubation at 33⁰C. The borrelia has been isolated from ticks, skin lesions, CSF, blood etc. Serological tests such as ELISA, immunofluorescence have been done. Antibodies take 1-2 months to appear, with initial IgM response, followed by IgG.                            Eventhough vaccines exists for humans, there are reports of damage by vaccine. Vaccines for dogs are available, they are 6 times more likely to develop the disease, cats have less chances because of their continual grooming habit. Doxycycline,  tetracyclines, Cephalosporins used in the treatment. Lyme disease can be controlled by avoiding tick bites. Since the tick is very tiny, it goes unnoticed. In tick infested areas, hat, long sleeves, long pants, socks should be worn. Tick repellent can be used. Once chronic Lyme disease is established, it crosses even the placenta and infect a foetus. Now a European wasp that parasitizes the ticks is being released in tick-infected areas(biological control). Also a protein, seen in the blood of lizards, is toxic to ticks feeding lizard's blood.

B. recurrentis : It causes relapsing fever, an arthropod borne infection, characterized by alternating fever and non-fever periods. Two types - Louse borne and Tick borne. Louse-borne infections are called epidemic relapsing fever by soft ticks Pediculus. Tick borne infections are called endemic relapsing fever by soft ticks Ornithodoros. Borrelia are microaerophilic, optimum temperature is 28-30°C. Cultivation in complex media with serum, also an allantoic membrane of chick embryos. They undergoes antigenic variations and this is the reason for relapses in the disease. This variation is due to DNA rearrangements in linear plasmids of Borrelia and finally body develop immunity to all variants.

Pathogenecity - After a tick/louse bite there is an incubation period of 2-10 days. After this RF sets in as fever. During this period borrelia are adundant in patient's blood. The fever subsides in 3-5 days.After 4-10 days, another fever sets in. Ultimately disease subsides after 3-10 relapses. Lice must be crushed and their body contents scratched into skin to transmit the disease. Ticks transmit organisms in their salivary secretions when biting and by transovarian transmission. The ticks survive 5 years without a meal and still contain live infections Borrelia. The disease is dangerous to pregnant women because the organism crosses the placenta and infect foetus.

Laboratory Diagnosis - A drop of blood may be examined under dark-ground microscope and borreliae detected by their lashing movements. Blood smears can be stained with Giemsa, Leishmania stain etc. A more successful method is to inoculate 1-2 ml of blood from the patient into white mice intraperitoneally. The borreliae multiply in the animals, occur in large numbers in the blood. Blood collected from tail vein and smear prepared, observation.

Prophylaxis and treatment - Louse borne RF occurs due to overcrowding and lack of personal hygeine. Tick borne RF occurs sporadically, associated with certain dwellings inhabited by ticks. No vaccine is available, insecticides can be used. Acute immunity seen. Tetracyclin, chloramphenicol used in the treatment.

Leptospira interrogans - They are actively motile, delicate spirochetes, with a large member of closely wound spirals and characteristics hooked ends. They are visualized under dark-ground illumination. They are saprophytic, some parasitic in rodents and other animals. Infection in general hosts is usually asymptomatic, but when other animals or human beings infected clinical disease result. 

Leptospires are grown in media enriched with rabbit serum. Semi-synthetic media-EMJH medium is used commonly. They are aerobic and microaerophilic. Optimum temp.is 25-30°C, optimum pH is 7.2-7.5. The generation time in media is 12-16 hours and 4-8 hours in inoculated animals. They may be grown in chorioallantoic membrane of chick embryos. 

  Leptospires are very sensitive to heat, being killed in 10 mins at 50°C and in 10 sec at 60°C, also sensitive to acid, gastric juice(30 mts), Bile, Chlorine, antiseptic, disinfectants. Their survival in water depends on temperature, acidity, salinity, nature of pollution. It survive for days in moist conditions at pH 6.8-8. It possess somatic antigens present in all members of the genus,surface antigens in serotypes.

Pathogenicity - L. interrogans causes Leptospirosis. It is a zoonosis, usually acquired by humans through contact with contaminated urine, enters the body through cuts or abrasions on the skin or through mouth mucosa, nose or conjunctiva. Dogs, cats and many wild animals carry these spirochetes. The bacteria live within the convoluated tubules of the kidneys and are shed into the urine. Often rain washes them from streets and the soil into natural bodies of water.Since the organism have a hook at the end, also borrow into the soft portions of the skin - usually soles of feet, palms of hands. After an incubation period of 10-12 days, it varies from mild to severe fatal illness. In mild cases, recovery occurs in 2-3 weeks. In severe cases, onset is acute with vomiting, headache and intense injection of eyes. A particularly virulent form of the infection, "Weil's syndrome", is characterized by jaundice and significant liver damage. They are seen in the blood during the acute phase of disease, but not after 8-10 days. They persist in the internal organs, abundantly in kidneys, therefore demonstrated in urine.

Laboratory Diagnosis - Diagnosis made by demonstration of leptospires microscopically in blood or urine, by isolating them in culture or by inoculation of guinea pigs or by serological tests

1. Examination of Blood: Blood examination is helpful only in the early stages of the disease, preferably before the administration of antibiotics. Demonstrated in blood under dark field microscope. 3 or 4 drops of blood are inoculated into each bottles containing EMJH medium, incubated at 37°C for 2 days and thereafter at room temperature in the dark for 2 weeks. Samples examined every third day for the presence of leptospires under dark background illumination. Leptospires sometimes isolated from the CSF also. The blood from the patient is also inoculated intraperitoneally into young guinea pigs. With virulent serotypes, animals develop fever and die within 8-12 days with jaundice and hemorrhage into the lungs and serous cavities. With non-virulent serotypes, they may not become ill and infection, identified by demonstration of leptospires in the peritoneal fluid, by blood culture or by serology.

2. Examination of urine: Leptospires appear in the urine in the second week of the disease and intermittently thereafter for 4-6 weeks.The urine should be examined immediately after collecting as leprospires undergoes lysis in acid urine. Centrifuged deposit of the urine may be examined under dark ground illumination.

3.Serological Diagnosis: Antibodies appear in serum towards the end of the first week of the disease and increase till the 4-th week,thereafter declining.Two types of serological tests:

1. Broadly reactive screening test

2. Type specific tests  

1. Broadly Reactive Sceening Test: It is also called as Genus specific test. Identify infection without indicating the exact infecting serovar. The antigens are prepared from non pathogenic L. biflexa. Also include agglutination, indirect immunofluorescence. ELISA used to detect IgM and IgG antibodies separately to indicate the stage of infection.

2. Type specific tests: This test identify the infecting serovar by demonstrating specific antibodies. Macroscopic Agglutination Test in which formalinised suspensions of prevalent leptospira serovars are tested with serial dilutions of the test serum. Microscopic Agglutination Test uses live cultures of different serotypes and agglutination is observed under the low power dark field microscope.

Prophylaxis ad treatment - Pathogenic leptospires survive for long periods in the convoluted tubules of the kidneys in natural hosts,multiply and are shed in the urine. Animal carriers often excrete 100 millions/ml of urine. If the infected urine contaminates water and humans come into contact with such water,leptospires enter the body through abraded skin or mucosa and initiate infection. The disease are common to agricultural workers. Rats are the prime carriers. So general measures include rodent control,water disinfection, wearing of protective clothing. Vaccination has been a success in dogs, cattles, pigs. Leptospires are sensitive to penicillin, tetracycline but the treatment to be effective should be started early in the course of disease.