Semisolid medium (0.2% agar) 

Korthof’s medium, Stuart’s medium, Fletcher’s medium (supplemented with inactivated rabbit serum) 

EMJH medium (supplemented with bovine serum albumin) 

Optimal temperature for growth (30ºC)

 

2.    Microscopic examination 

 

Dark-field microscopy (blood/urine) 

‘hook-like end’ is a characteristic morphology feature. 

Actively motile, rotating steadily around their long axis.

 

3.       Serological examination

Demonstration of the antibody against leptospires 

1. Microscopic agglutination test (MAT) 

2. Enzyme-linked immunosorbent assay (ELISA)

 

 

Antibody response

 What is the nature of the antibody response?

 Humans react to an infection by leptospires by producing specific anti-Leptospira antibodies. Seroconversion may occur as early as 5–7 days after the onset of disease but sometimes only after 10 days or longer. IgM class antibodies usually appear somewhat earlier than IgG class antibodies, and generally remain detectable for months or even years but at low titre.

 

 MICROBIOLOGY AND IMMNUNOLOGY 

 Antibody response Detection of IgG antibodies is more variable. They may sometimes not be detected at all, or be detectable for only relatively short periods of time, but may sometimes persist for several years. The antibodies are directed against (Annex 8): – common antigens (so-called genus-specific antigens) that are shared by all leptospires, both pathogenic and saprophytic; – serovar-specific and serogroup-specific antigens. Patients with leptospirosis may produce antibodies that react with several serovars. This phenomenon, called cross-reaction, is often observed in the initial phase of the disease. After the acute disease, cross-reactive antibodies gradually disappear as the immune response "matures", usually in the course of weeks or months, while serogroup- and serovar-specific antibodies often persist for years. Thus the genus-specific antibodies usually remain detectable for months, the serovarspecific antibodies for years. Weak cross-reactions may also occur to other groups of microorganisms and will vary with the serological method used. Occasionally patients produce specific antibodies that react with broadly reactive antigens only, and few or no serovar-specific antibodies are detected. Some other patients may produce only serovar-specific antibodies. Protective immunity Is the antibody response protective? It is generally believed that serovar-specific antibodies are protective and that a patient is immune to reinfection with the same serovar as long as the concentration (titre) of specific antibodies is high enough. Antibodies provoked by an infection with a particular serovar do not necessarily protect against infection with other serovars.

 

Serological tests 

What methods are used for serodiagnosis? 

The microscopic agglutination test (MAT) is considered the "gold standard" or cornerstone of serodiagnosis because of its unsurpassed diagnostic (serovar/serogroup) specificity in comparison with other currently available tests. A variety of other serological methods, including the enzyme-linked immunosorbent assay (ELISA) have been developed, many of which are relatively simple screening tests for leptospirosis. 

 

MAT 

What is the microscopic agglutination test (MAT)? The MAT is a test which determines agglutinating antibodies in the serum of a patient by mixing it in various dilutions with live or killed, formolized leptospires. Antileptospiral antibodies present in the serum cause leptospires to stick together to form clumps. This clumping process is called agglutination and is observed using dark-field microscopy. Agglutinating antibodies can be of both IgM and IgG classes. 

 

MAT specificity

How is the specificity of the MAT shown? Patients usually produce agglutinating antibodies against the infecting serovar.  However, antibodies that cross-react with other serovars are also often found, and this is particularly noticeable early in the course of the infection. In the first few weeks of the disease, heterologous cross-reactions with other serovars may be even stronger than the homologous reaction with the infective serovar. Occasionally, a heterologous reaction can be positive while the homologous reaction is or remains negative, a phenomenon called a paradoxical reaction. The titres of cross-reactive antibodies tend to decrease relatively quickly, after months, while serogroup- and serovar-specific antibodies may persist for a much longer time, often for years. It has been found that: (a) agglutinating antibodies often react only with a certain serovar or serogroup; (b) many serovars may circulate and cause disease in a given area; (c) the prevalence of different serovars may change as a result the of introduction of new maintenance hosts, agricultural practices, etc; (d) new serovars may be introduced. For this reason panels of live leptospires belonging to different serovars must be maintained in the laboratory to be used as antigens in the MAT. These panels should include, as a minimum, all locally occurring serovars. If the panel is incomplete, antibodies to the serovar that is missing from the panel may not be detected and serodiagnosis may give inaccurate or false-negative results. If the locally circulating serovars are not known or subject to change, the panel should consist of serovars representing all serogroups. 

 

MAT titres 

When is a MAT titre positive or significant? The MAT cannot differentiate between agglutinating antibodies due to current, recent or past infections. Ideally, as with other serological tests, two consecutive serum samples should be examined to look for seroconversion or a four-fold or greater rise in titre. 

 

MAT: advantages and disadvantages 

What are the advantages and disadvantages of the MAT? The major advantage of the MAT is its high specificity. An important disadvantage is the need for facilities to culture and maintain panels of live leptospires. Furthermore, the test is both technically demanding and time consuming, particularly when the panel is large. An obvious but definite shortcoming is that antibodies may not be detectable when the causative strain is not represented in the panel or only a low titre is found with a serovar that antigenically resembles the absent causative serovar. The finding of no titre or a low titre in the MAT does not exclude leptospirosis in these circumstances. It is never possible to be sure that the panel is complete since new, unidentified leptospires may cause disease. For this reason it is advisable to include a genusspecific screening test such as an ELISA using a broadly reactive antigen. 

 

Serovar-/ serogroup-specific agglutination 

Does serology reveal the causative serovar in the individual patient? Patients usually produce agglutinating antibodies against the infecting serovar. In patients with current or recent infection, cross-reactivity in the MAT may make it impossible to identify the infecting serovar or its serogroup with any degree of certainty. It may be useful to collect a serum sample some time (e.g. 1 month) after the onset of the disease in the hope that, by that time, the residual antibodies will be sufficiently specific to give an indication of the serogroup or, more rarely, the serovar. 

 

ELISAs usually only detect antibodies reacting with a broadly reactive genus-specific antigen and thus give no indication of the causative serovar or serogroup. Seroepidemiology Does serology reveal the range of serovars that cause infection in a population in a certain area? The MAT may reveal the presumptive serogroup to which the causative serovar may belong based on agglutination with different serovars in a panel representative of the locally circulating serovars. Under the best conditions, it may reveal the serovar. Thus seroepidemiological investigations of serum samples from the general population may indicate circulating serogroups since residual antibodies from past infections tend to react with serogroup-specific antigens. 

 

ELISA / Other (commercial) tests 

Which other serodiagnostic methods other than the MAT are often used? 

ELISAs are popular and several assays are available. They can be performed with commercial kits or with antigen produced "in house". A broadly reactive so-called genus-specific antigen is generally used to detect IgM, and sometimes also IgG antibodies. The presence of IgM antibodies may indicate current or recent leptospirosis, but it should be remembered that IgM-class antibodies may remain detectable for several years. 

 

ELISA: advantages and disadvantages 

What are the advantages and disadvantages of the ELISA in comparison with the MAT? Advantages: · ELISA can detect IgM-class antibody in the early phase of the disease so that current or recent infection may be indicated. Where no antibody is detected or only a low ELISA titre is found, a second serum sample should be examined for seroconversion or a significant rise in titre. · Only a single antigen is used, namely the genus-specific antigen, which is shared by pathogenic and saprophytic leptospires alike. · In contrast to the MAT, ELISA can be standardized. · Culture of leptospires in the local laboratory to provide the antigen is not required if a commercial source of kits is available. 

Disadvantages: · Some ELISA test systems are less specific than the MAT and weak crossreactions due to the presence of other diseases may be observed. ELISA results should therefore be confirmed by the MAT. This may require testing a follow-up sample if the initial sample was taken at an early stage in the infection when the ELISA test may be positive, but the MAT negative. · Since it is based on a genus-specific antigen, the ELISA test does not give an indication of the infecting serovar.

- In both Hong Kong, US and UK, dogs reported to contact or die from Leptospirosis even after vaccination 

- Ineffectiveness of vaccine due to high number of serovars and strains that are not covered by vaccine (do not provide cross-protection)

- Vaccinated animals may shed the Leptospira microbes and become the source of infection for owners (Duck hunter in California came down with Leptospirosis and the water sources he frequented are found to be contaminated, most probably by his vaccinated dog).

Leptospirosis Vaccine Side Effect
-          Antigen for Leptospira vaccine create the disease manifestation 

-          Dogs may die of renal failure within 48 hours after vaccination

-          Development of tumors, mast cell disease and cytokine storms (an overreaction of the immune system where too many immune cells are activated at single location) which in turn creates a potential damage to body tissues and organs

-          Autoimmune disease induced by Leptospira vaccine especially those that comes with aluminium adjuvant (carcinogenic) 

-          Unacceptable side effects

-          Incomplete and short lived protection

-          Increased risk of developing autoimmune disease

-          Vaccination that only provides partial protection will cause the disease to be spread in the environment (where the organism can be found in urine)