Leptospira

Biology

Leptospirosis – caused by a bacteria of the genus Leptospira that affects humans and animals. It can cause a wide range of symptoms, some of which may be mistaken for (other diseases particularly in humans. However, some infected persons, may not exhibit symptoms at all. (Centers for Disease Control and Prevention, 2019) Virtually all mammals are affected by this disease and has a broad range of clinical effects, from mild, subclinical infection to multiple-organ failure and death. (Lunn, 2019)

Classification

According to Kmety et al (2003) and Plank et al (2000), traditional classification specified that all saprophytic species are under Leptospira biflexa, while L. interrogans included all pathogenic species of leptospires. The currently used genetically based classification indicates that there are at least 19 species (13 pathogenic and six saprophytic (Adler et al, 2010 and Bharti et al 2003)), identified through DNA hybridization analysis (Brenner et al, 1999 and Levett, 2001). Seven of these species: L. interrogans, L. borgpetersenii, L. santarosai, L. noguchii, L. weilli, L. kirschneri and L. alexanderi are the main agents of leptospirosis (Ahmed et al, 2006).

Cell Biology

All recognized species of Leptospira are categorized into 24 serogroups and 250 serovars (Palanniapan et al, 2007), based on the expression of surface-exposed lipopolysaccharide (LPS) (Adler et al, 2010). The determinant of antigenic diversity among serovar groups is their structural differences in the carbohydrate moiety of LPS determines antigenic diversity among the numerous serovar groups. Serovars containing overlapping antigenic determinants are classified into a larger serogroup. Phylogenetic analyses of 16S rRNA genes suggest that Leptospira species cluster into three groups designated pathogenic, saprophytic and intermediate (Levett et al, 2006 and Perolat et al, 1998).

Usually 6-20 μm in length, leptospires are thin, helically coiled, motile spirochetes. Its distinctive question-mark shape at the hooked ends of this bacterium makes it easier to identify. The leptospires have surface structures that share features of both Gram-positive and negative bacteria. The double-membrane and the presence of LPS are characteristic of Gram-negative bacteria, while the close association of the cytoplasmic membrane with murein cell wall is reminiscent of Gram-positive envelope architecture (Levett, 2001, Haake, 2000, Ko et al 2009 and Vijayachari et al, 2008).

The two periplasmic flagella or endoflagella functions in motility which arise from each end of the bacterium. The disruption of the flagellin gene flaB by a kanamycin marker in saprophytic L. biflexa through homologous recombination resulted in the absence of the endoflagella with the corresponding loss in bacterial movement (Charon et al, 2002, Picardeau et al, 2001 and Wolgemuth et al, 2006). On the other hand, in both liquid and semi-solid media, the flagellar motor switch fliY mutant of the pathogenic L. interrogans exhibited attenuated rotative motion (Liao et al, 2009). Guinea pigs that are also infected with the fliY mutant had a higher survival rate compared with those infected with wild-type strains, suggesting that endoflagellar rotation and consequent bacterial motility might have roles in the pathogenesis of Leptospira infection.

Epidemiology

According to World Health Organization, leptospirosis is a major, yet under recognized threat to public health. This bacterial disease causes morbidity and mortality around the world. Although it is endemic in many rural and urban slum communities and can also cause sporadic epidemics, little is actually known about the true disease burden and consequently, the disease has been neglected.

Leptospirosis in wildlife is common, although the disease is most often noticed only when the wildlife serve as a source of infection for domestic animals or people. Leptospirosis is found throughout the world. The infection (and disease) is more prevalent in warm, moist climates and is endemic in much of the tropics. The disease is more seasonal, with the highest incidence after periods of rainfall in temperate climates. (Lunn, 2019)

In 2009, there were 2,158 cases of leptospirosis, 167 of which are death cases due to flooding in Metro Manila. (Berger, Gideon Informatics, 2019) 

In the Philippines, leptospirosis is highly endemic. Outbreaks usually occur during the typhoon season (July–October) (Victoriano, 2009, Yanagihara, 2007 and Pappas, 2008). On September 26, 2009, a typhoon caused serious flooding in Metro Manila. The number of patients with suspected signs and symptoms of leptospirosis increased sharply starting in the first week of October (Republic of the Philippines, National Disaster Coordinating Council Situation report No. 52). 2,299 patients, including 178 who died (case-fatality ratio [CFR] 8%), in 15 hospitals in Metro Manila until mid-November were reported to the Department of Health. Amilasan et. al in 2009 conducted a hospital-based investigation to describe the characteristics of hospitalized patients, investigate risk factors for death, and identify the causative Leptospira species and serogroups.

The Department of Health released a report from January – March 2018, a total of 494 leptospira cases in the Philippines nationwide. In their geographic distribution, Regions 6 (25.9%) , 9 (15.2%) , 11 (13.6%), NCR (7.7%) and Region 8 (6.3%) has the most number of cases.

Pathogenesis

After penetrating exposed mucous membranes or damaged skin, leptospires invade the body. Usually after a variable incubation period (4–20 days), leptospires circulate in the blood and replicate in many tissues including the liver, kidneys, lungs, genital tract, and CNS for 7–10 days. The clinical signs of acute leptospirosis, which vary by serovar and host, occur during the period of bacteremia and tissue colonization. Soon after leptospiremia occurs and coincide with clearance of the leptospires from blood and most organs, agglutinating antibodies can be detected in serum as the organisms are cleared, the clinical signs of acute leptospirosis begin to resolve, although damaged organs may take some time to return to normal function. In some cases, severely damaged organs may not recover, leading to chronic disease or death.

At this point, in incidental and maintenance hosts, the disease diverges. Leptospires remain in the tubules of the kidneys of incidental hosts for a short period of time and are shed in the urine for a few days to several weeks. However, leptospires often remain in the renal tubules, genital tract, and less commonly, the eyes, despite the presence of high levels of serum antibody in maintenance hosts. After initial infection, leptospires are shed in the urine and genital secretions of persistently infected animals for months to years and these animals become an important reservoir of infection, with the potential to transmit infection to other reservoir hosts or to incidental hosts at risk of developing clinical disease. (Lunn, 2019)

Diagnosis and Prevention

Dr. Katherine Lunn enumerated several tests in diagnosing leptospirosis. In order to have a good grasp of the diagnosis of leptospirosis, one must have a good clinical and vaccination history and laboratory testing. Diagnostic tests for leptospirosis include those designed to detect antibodies against the organism and those designed to detect the organism in tissues or body fluids. In each case, serologic testing is recommended combined with one or more techniques to identify the organism in tissue or body fluids.

The most commonly used technique to diagnose leptospirosis in animals are serologic assays. 

Microscopic Agglutination Test (MAT)

The is the most frequently used technique. It involves mixing appropriate dilutions of serum with live leptospires of serovars prevalent within the region. The presence of antibodies is indicated by the agglutination of the leptospires, with the reported titer being the highest dilution of serum that results in 50% agglutination. The MAT is a complex test to perform and interpret, and it requires the maintenance of live leptospiral cultures. 

ELISA (Enzyme-Linked Immunosorbent Assay)

A commercial laboratory in the US uses ELISA test to diagnose canine leptospirosis. This test detects antibodies to LipL32, a membrane protein found on pathogenic leptospires. The currently available assay provides a qualitative negative or positive result and will also detect antibodies induced by vaccination. A comparison of this test to the MAT has not been reported, and it is likely that the numerical titers provided by the MAT will provide more diagnostically useful information than a qualitative ELISA.

Interpretation of serologic results from the MAT is complicated by a number of factors, including cross-reactivity of antibodies, antibody titers induced by vaccination, and lack of consensus about the level of antibody titer that indicates infection. Antibodies produced in an animal in response to infection with a given serovar of Leptospira often cross-react with other serovars. In some cases, these patterns of cross-reactivity are predictable based on the antigenic relatedness of the various serovars of Leptospira, but the patterns of cross-reactive antibodies vary between host species. Paradoxical reactions may occur with the MAT early in the course of an acute infection, with a marked agglutinating antibody response to a serovar other than the infecting serovar. In addition, there is evidence of lack of consistency between diagnostic laboratories. For these reasons, the infecting serovar in an individual animal cannot be reliably identified as the serovar to which the animal develops the highest titer. The real value of the MAT is in providing a numerical titer to allow comparison of acute and convalescent values.

As to what constitutes a diagnostic titer for leptospiral infection, a consensus is lacking. Because titers are often low in acute disease and in maintenance host infections, a low antibody titer does not necessarily exclude a diagnosis of leptospirosis. A 4-fold rise in antibody titer is often observed in paired serum samples collected 7–10 days apart in cases of acute leptospirosis. Diagnosis of leptospirosis based on a single serum sample should be made with caution and with full consideration of the clinical picture and vaccination history of the animal. In general, with a compatible clinical history and vaccination >3 mo ago, a titer of 1:800 to 1:1,600 is good presumptive evidence of leptospiral infection. The use of paired acute and convalescent titers is strongly recommended whenever possible. Antibody titers can persist for several months after infection and recovery, although there is usually a gradual decline with time.

Immunofluorescence

Immunofluorescence can be used to identify leptospires in tissues, blood, or urine sediment. Although the test is fast and has a reliable sensitivity, the interpretation requires a skilled laboratory technician. Immunohistochemistry is useful to identify leptospires in formalin-fixed tissue but, because there may be small numbers of organisms present in some tissues, the sensitivity of this technique is variable. 

Polymerase Chain Reaction (PCR)

A number of PCR procedures are available, and each laboratory may select a slightly different procedure. PCR techniques allow detection of pathogenic leptospires in blood, urine, or tissue samples but do not determine the infecting serovar. Numerous PCR assays has been developed due to the need for rapid diagnostics at the time of admission. While treatment may be effective, their advantage lies in the ability to obtain a definitive diagnosis during the acute stage of the illness prior to antibodies are detectable. (Lunn, 2019).

In the first 5–10 days after the onset of the disease and up to the 15th day, PCR can already detect DNA in the blood. The bacterial load in serum/blood ranges from 105 to 109 leptospires/L. PCR allows detection of leptospires in culture negative blood if the patient has received an effective antimicrobial drug but have not cleared nonviable organism. (Musso and La Scola, 2013)

Transmission

It is most commonly spread via water contaminated with urine from infected animals, but contaminated food or soil can also act as vehicles for the disease. The main animal reservoirs are rodents, livestock and dogs. Disease in humans can vary from mild flu-like illness to serious disease. Some severe complications include kidney damage, liver failure, respiratory distress, meningitis and death. (World Health Organization, 2019)

Zoonosis

According to Centers for Disease Control (2019), leptospirosis occurs worldwide, but is most common in temperate or tropical climates. It is an occupational hazard for many people who work outdoors or with animals, such as:

• Farmers
• Mine workers
• Sewer workers
• Slaughterhouse workers
• Veterinarians and animal caretakers
• Fish workers
• Dairy farmers
• Military personnel

With most of the pathogenic serovars of Leptospira, humans are susceptible to infection but are incidental hosts and, therefore, not important reservoirs of infection. As mentioned above, occupational exposure is a risk factor, and veterinarians, veterinary staff, livestock producers, and dairy workers are at increased risk. In addition, recreational exposure to waters contaminated with urine of domestic animals or wildlife presents a risk. Animal owners have contracted leptospirosis via contact with infected companion animals and livestock.

The principal route of infection is contact with infectious body fluids (blood in acute cases or urine) via mucous membranes. The disease varies from subclinical to severe and can be fatal when renal or hepatic failure occurs in humans. The most common signs are fever, headaches, rash, ocular pain, myalgia, and malaise. Transplacental infection, abortion, and infection of infants via breast feeding have been described, making exposure of pregnant women of particular concern. Laboratory techniques are necessary for a definitive diagnosis. (Lunn, 2019)

Precaution

The diagnosis of leptospirosis in animals can be difficult with clinical signs alone, therefore veterinarians may wish to implement an infection control program in which animal body fluids are handled only with gloved hands and hand washing is routine. It is also essential for staff to take precautions when handling or nursing animals suspected or confirmed to have leptospirosis. Wearing gowns, shoe covers, and gloves to avoid contaminating exposed skin or spreading organisms are the appropriate precautions. To avoid contact of aerosolized organisms with mucous membranes, face shields should be worn when handling wet bedding or cleaning cages, stalls, or runs.

Risk Factors

Rodents and fresh water are the primary risk factors. The bacteria causing leptospirosis can survive for up to six months in fresh water or a humid location once dispersed through the urine of contaminated animals, such as rats or livestock. (Ferguson, 1994 and Andre-Fontaine G et. al, 2015). It is essential that those who are more at risk of exposure should take measures to protect themselves, as Leptospira are thin, mobile bacteria that can penetrate the body through various means (Adler B., 2015). The point of entry of this bacteria are: wounds or scratches, eyes, nose, mouth, dilated pores of healthy macerated skin.

According to American Veterinary Medicine Association, dogs are most commonly affected by leptospira. Although very little is known about the disease in cats, leptospirosis in cats is rare and appears to be mild. Common risk factors for leptospirosis in dogs residing in the United States include exposure to or drinking from rivers, lakes or streams; roaming on rural properties (because of exposure to potentially infected wildlife, farm animals, or water sources); exposure to wild animal or farm animal species, even if in the backyard; and contact with rodents or other dogs.

Dogs can become infected and develop leptospirosis if their mucous membranes (or skin with any wound, such as a cut or scrape) come into contact with infected urine, urine-contaminated soil, water, food or bedding; through a bite from an infected animal; by eating infected tissues or carcasses; and rarely, through breeding. It can also be passed through the placenta from the mother dog to the puppies.