Leptospirosis: Its Epidemiology, Diagnosis and Control

Leptospirosis is a zoonosis caused by pathogenic spirochetes of the genus Leptospira.
Disease was first described by Adolf Weil in 1886
In 1908, a Japanese research group led by Ryokichi Inada and Yutaka to first identified the bacterium as the causative agent of leptospirosis and noted its presence in rats in 1916
Generally it is transmitted by the infected urine of rodents.
Leptospirosis is in the group of 17 neglected tropical diseases, categorized by WHO.
Leptospirosis is an underreported disease, and there are no reliable global incidence figures (WHO, 2015)
Synonyms: Weil's Syndrome, Weil-Vasiliev disease, Swineherd's disease, Rice-field fever, Waterborne fever, Nanukayami fever, Cane-cutter fever, Swamp fever, Mud fever, Stuttgart disease, and Canicola fever.

1. Leptospirosis: Its Epidemiology, Diagnosis and Control with particular reference to Assam

2. INTRODUCTION Leptospirosis is a zoonosis caused by pathogenic spirochetes of the genus Leptospira . Disease was first described by Adolf Weil in 1886 In 1908, a Japanese research group led by Ryokichi Inada and Yutaka to first identified the bacterium as the causative agent of leptospirosis and noted its presence in rats in 1916 Generally it is transmitted by the infected urine of rodents. Leptospirosis is in the group of 17 neglected tropical diseases, categorized by WHO. Leptospirosis is an underreported disease, and there are no reliable global incidence figures (WHO, 2015) Synonyms : Weil's Syndrome , Weil- Vasiliev disease, Swineherd's disease, Rice-field fever, Waterborne fever, Nanukayami fever, Cane-cutter fever, Swamp fever, Mud fever, Stuttgart disease, and Canicola fever.

3. Etiology Aerobic, gram-negative, slow growing Spirochetes that are fastidious Characteristic corkscrew-like motility Leptospira  were divided into two groups; The pathogenic  Leptospira  were all classified as members of  L. interrogans The Saprophytic   Leptospira  were classified as  L biflexa Long, Thin, Highly Coiled Under Dark Field Microscopy Source: Merck Veterinary Manual Source: Wikipedia

4. Table: Various serovars and their respective carriers

5. CURRENT STATUS OF THE DISEASE Worldwide major outbreaks were reported from the Latin America and the Caribbean region (36%), followed by Southern Asia (13%), and North America (11%) (Claudia et. al ., 2020) In India, the disease is prevalent in almost all the species of domestic animals and in several rodent species It has been reported more frequently from southern states although few reports are also available from western states (Gujarat and Rajasthan) (WHO, 2015) Human Leptospirosis is reported to be of endemic importance from many States of India which includes Andaman and Nicobar Islands (since 1988), Gujarat (since 1994), Maharashtra ( since 1998), Kerala and Tamil nadu . (WHO, 2015) Few reports from Assam. Kader et.al ., 2018 carried out a study in cattle from few districts of lower Brahmaputra valley, Assam and recorded a seropositivity of 11.58 %.

6. Three epidemiological patterns of leptospirosis were defined by Faine (1994) The first occurs in temperate climates where few serovars are involved and human infection almost invariably occurs by direct contact with infected animals though farming of cattle and pigs The second occurs in tropical wet areas , within which there are many more serovars infecting humans and animals and larger numbers of reservoir species, including rodents, farm animals, and dogs The third pattern comprises rodent-borne infection in the urban environment (slums) EPIDEMIOLOGY

7. EPIDEMIOLOGY HOST RANGE   1. Reservoir host: wide range of natural rodent and non-rodent reservoir hosts which include foxes, rabbits, rat, mice, moles etc.   2. Carrier hosts : Domestic animals includes cattle, sheep, goat, buffalo, horse and dogs.  3. Accidental/ Incidental host : Human act as accidental host of disease. MODE OF TRANSMISSION 1. Direct contact with an infected animal. No human to human transmission is reported 2. Indirect contact via soil, water and food contaminated with urine from an infected animal 3. Transplacental transmission may occur if infection occurs during pregnancy  4. Droplet infection –breathing air polluted with droplets of urine, eg . During milking of cattle and buffalo.

8. FACTORS FAVOURS EMERGENCE OF LEPTOSPIROSIS Reservoir and carrier host Soil salinization -The salinity of the soil provides favourable environment for survival of leptospires for months together Climate change - These factors include rainfall, humidity, ambient temperature, water retaining capacity of the soil, pH and salinity of the soil and surface waters and forest cover Seasonal variation - It is usually a seasonal disease that starts at the onset of the rainy season and declines as the rains recede. Sporadic cases may occur throughout the year Rodent density Population size of the farm and other domestic animals Sanitation of animal habitats Availability of veterinary/ medical services for prompt detection and treatment of animal leptospirosis and control programs Personal hygiene

9. HIGH RISK GROUPS ● Occupational exposure – Farmers, ranchers, abattoir workers, trappers, veterinarians, loggers, sewer workers, rice farmers, pet traders, military personnel, laboratory workers ● Recreational activities – Freshwater swimming, canoeing, kayaking, trail biking ● Household exposure – Pet dogs, domesticated livestock, rainwater catchment systems, infestation by infected rodents ● Other – Walking barefoot through surface water, skin lesions, contact with wild rodents, accidental laboratory exposure

10. Occupational vulnerability: about 75% of leptospirosis cases are farmers Recreational vulnerability affecting a wider range of rural populations Leptospira interrogans , transported by water in the environment Vectors of leptospirosis: Rice field rodents ( Bandicota sp. And Rattus sp.) Leptospirosis: a Zoonoses amplified by seasonal flooding

11. Transmission cycle and pathogenesis of leptospirosis  Faisal et. al., 2012 Leptospires invade the body Via penetrating exposed mucous membranes or damaged skin After a variable incubation period (4–20 days) leptospires circulate in the blood replicate in many tissues including the liver, kidneys, lungs, genital tract, and CNS leading to various manifestation

12. DISEASES IN ANIMALS In Cattle : Incubation period (I.P) :  2–12 weeks Acute form : Sudden onset of elevated temperature (40-41.1°C), anorexia, anaemia /jaundice, hemoglobinuria, abortion and depression may be seen Subacute form : Onset is slow, 'milk drop syndrome ' due to mastitis resulting in reduced yield, thick flaking like of colostrum) and yellow to blood coloration of milk is common. The symptoms lasts for about weeks. Jaundice may be seen in some cases Chronic form : Abortions, still births, foetal deaths, weak calves and retained placenta cases are common in herds.

13. DISEASES IN ANIMALS Dogs: I.P:  5–15 days Elevated body temperature, depression, deep sunken eyes , anorexia, muscle tenderness, vomiting , intussuseption , foul breath, ulcerated gums are usually associated with anaemia and extensive jaundice that may lead to death. The acute form of disease, known as “ Stuttgart disease” characterized by vomiting, rapid dehydration, collapse, necrosis and sloughing of buccal mucosa and tongue Image depicting ulcerated gums and sunken eyes Image depicting vomition

14. DISEASES IN ANIMALS Swine: Usually infection is mild or inapparent . Occasionally may show pyrexia. Only or few affected pigs show acute signs of anorexia, pyrexia and/ or diarrhoea for 1-3 days. Chronic cases usually abort in late pregnancy or give birth to weak piglets . and Recover become renal shedders for at least 6 months. Horses : Disease manifestations, though rare and usually mild, include pyrexia, icterus, periodic ophthalmic and abortion. Sheep: The common signs include abortion, fever, agalactia, dysponea , jaundice, hematuria and sudden death. Goats: May remain symptomless but shed leptospires in the urine for short periods or may develop jaundice, haemaglobinurea and abortions. Cats: Although susceptible to experimental infection, seldom suffer from clinical leptospirosis.

15. DISEASES IN MAN After an incubation period of 7-14 days the disease is manifested in 2 phases . Early or leptospiraemic phase: The pathogen appears in blood and CSF. In milder cases, the disease remains a subclinical infection In others, it may present itself as a 'flu-like' syndrome which is characterized by sudden high fever, chills, headache, muscle aches, vomiting and conjunctivitis. Non availability of treatment could intensify the disease to second phase. 

16. 2 . Second or immune phase/Well's syndrome: The symptoms may gradually worsen leading to kidney and liver failure , of which the latter results in jaundice Widespread haemorrhages occur leading to anaemia , coma and finally death. Fig: Red eye caused by leptospirosis Red eye (conjunctival suffusion due to immune reaction ) is a constant and characteristic feature. In rare cases , the most severe forms of the disease present as atypical pneumonia, or aseptic (leptospiral) meningitis or myocarditis All the severe forms have a mortality rate of 5-10%, which is quite significant

17. Diagnosis The disease can be tentatively diagnosed on the basis of clinical symptoms However, confirmatory diagnosis is made as follows: Direct examination of samples : The clinical or morbid materials such as blood, urine, CSF, tissue scrapings/emulsions should be properly treated with 10% acetic acid for 5-19 minutes (in case of impression smears), or 0.25% trypsin for 3-5 minutes (in case of tissues), or 40% formalin to decontaminate clinical materials, or in smears by following microscopic techniques as Dark field microscopy at 400x magnification Silver impregnation staining Silver impregnation staining for Leptospira

18. Isolation of leptospires : These methods include ( i ) Culture, (ii) Animal inoculation, and (iii) Culture following animal inoculation (ii) Animal inoculation: Carried out to enhance the concentration of Leptospira cells or to remove the bacterial contaminants from the samples. In hamsters (3 weeks old): Fatal infection accompanied by jaundice evoked following inoculation with 0.5-1 ml of sample through intra-peritoneal route Guinea pigs (1 week old) inoculated with 0.5-1 ml material, i /p seldom die of the infection ( i ) Culture: The isolation rate of leptospires from clinical specimens is very low. Media for culturing leptospires are of 3 types, media containing serum ( Korthof's medium, Fletcher's semi-solid medium etc.) media containing bovine albumin fraction V and tween 80 (EM medium, EMJH etc ), and synthetic media

19.   Other test being employed:   Immunofluorescence: Useful in examination of urine, other body fluids, and tissues that have been frozen or are not amenable to silver staining.   Immunohistochemistry: Achieved by using enzymatic or metallic labels on the secondary antibody. Phosphatase, peroxidase, or metallic gold- labelled antibody can be used.   Nucleic Acid Probes and Hybridization: Leptospira specific sequences are isolated, cleaved and labelled with a reporter molecule , then hybridized to ss DNA . If the nucleotide sequences in the nucleic acid probe are complementary to those in the sample, hybridization occurs monitored by autoradiography or calorimetrically   Polymerase Chain Reaction : PCR can rapidly confirm the diagnosis in the early phase of the disease, when bacteria may be present and before antibody titres are at detectable levels.

20. SEROGROUP/SEROVAR SPECIFIC TEST: Microscopic Agglutination Test (MAT): MAT is the WHO standard reference test for serological diagnosis of leptospirosis . MAT determines agglutinating antibodies in the serum of a patient/ animal by mixing it in various dilutions with live/killed leptospires . Anti-leptospiral antibodies present in the serum cause the leptospires to stick together to form clumps observed using dark field microscopy (DFM). Accepted endpoint: Final dilution of serum at which 50% or more of the leptospires are agglutinated. Leptospires observed under DFM (20X)

21. GENUS SPECIFIC TESTS: These tests are based upon the use of a single antigen common for the genus Leptospira . The antigen for these tests is prepared from the non-pathogenic L. biflexa Patoc –1 strain. Macroscopic Slide Agglutination Test: This test is carried out with a dense suspension of leptospires , which agglutinate into clumps visible to the naked eye. Indirect Fluorescent Antibody Test: Specimens of blood, urine and parenchymatous organs are stained with luminescent sera and examined under a fluorescent microscope. The antigen antibody complex fluoresces brightly and is visible under the microscope.   Complement Fixation Test (CFT): CFT is performed using either whole leptospiral cells or soluble extracts. The CFT is useful in detecting relatively recent infection. .

22. Enzyme Linked Immuno Sorbent Assay (ELISA): ELISA usually detects only the antibodies reacting with a broadly reactive genus-specific antigen. Microcapsule Agglutination Test (MCAT): This test is based on the passive agglutination of synthetic polymer carriers, sensitized with mixed antigens of sonicated leptospires , by leptospiral antibody. Lepto Dipstick: This dipstick assay for the detection of Leptospira-specific IgM antibodies in sera Latex Agglutination Test: This test depends on the sensitisation of commercially available latex particles with a leptospiral antigen.

23. CONTROL STRATEGIES IN ANIMALS:   1.Rodent control: Areas which favour rodent survival, the rodent control should be exercised by suitable means including ( i ) poisoning with rodenticide (2% zinc phosphide) , (ii) use of predators (cat, cagle etc.), (iii) trapping, (iv) biological control either by manipulating or altering the habitat, (v) mechanical proofing, (vi) rodent repellents, (vii) electronic barriers (fencing), (viii) fumigation, (ix) proper storage of foods, animal feeds and grains, and (x) general cleanliness of surroundings

24. 2. Sanitation: ( i ) proper draining of yards, pens, sheds and kennels and their regular disinfection (with cresol , sodium hypochlorite etc.), (ii) avoidance of stagnation of water, urine and faeces, and (iii) burial or burning of infected carcasses, aborted foetus , placentas and/or bedding   3. Proper Management: Should aim at ( i ) breaking of the cycle of infection (ii) allowing new addition to herd or flock only if such animals have been found negative to MAT on their initial testing as well on their retesting sero -testing of herd by MAT strictly remove carrier animals serving as excreters and to isolate infected animals (iv) prevention of reinfection

25. IN MAN: The disease control measures include 1. Personal hygiene and protection emphasising in ( i ) protection of food articles and utensils from contamination with urine of rat (ii) encouraging use of protective clothing (rubber gloves, goggles, gum-boots particularly when working in water logged areas or handling animals/animal products during slaughtering and parturition etc., (iii) avoidance of swimming and wading in water of lakes, ponds, swimming pods contaminated with urine of rats and livestock, and (iv) encourage mechanization of agricultural operations.

26. 2. Sanitation: It includes ( i ) disinfection of contaminated work areas such as food stores, abattoirs, fish and meat processing plants and animal sheds, (ii) proper collection, transport, treatment and secured disposal of garbage and animal excreta (iii) proper disposal of dead and infected animals, (iv) disinfection of swimming pool with chlorine, (v) drainage of wet areas, and (vi) rodent control in the areas of domestic and farm environment.

27. 3. Animal related care : It calls for ( i ) care in handling of laboratory and other animals as they may be carriers, (ii) improvement in occupational hygiene standards in animal farms/ laboratories   4. Health education : General people and particularly the high-risk group groups should be educated about the disease, and the protective measures to be followed including prohibition of activities in contaminated waters.

28. CONCLUSION Leptospirosis is a worldwide underreported neglected zoonosis In many developing countries, including most of the leptospirosis endemic areas, laboratory capabilities to detect pathogenic microorganisms are often inadequate Reservoirs control measures, environmental control programs and animal vaccination, in combination with a strong surveillance system may significantly reduce the disease Public education for people’s awareness of the disease reservoirs and its transmission is essential The extensive and good understanding of the eco-epidemiological and cultural characteristics of a community that faces the problem of leptospirosis is an essential prerequisite for evolving an effective and acceptable control measure.

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