Method for identifying bacteria of the genus leptospira

Abstract

A method for identifying affiliation of Leptospira isolates to a specific serogroup and serovar using analysis of the percentage composition of monosaccharides in LPS of Leptospira was disclosed, it allows the identification of the affiliation of the tested strains without the use of antibody techniques, which reduces the costs of diagnosis and allows it to be carried out in less specialized research units. The method can find a particular use in veterinary diagnostics and diagnostics of zoonoses.

Description

[0001] Method for identifying bacteria of the genus Leptospira

[0002] The object of the invention is a method for determining the serovar affiliation of bacterial strains belonging to the genus Leptospira. The method of the invention may be commonly used in research and diagnostic laboratories characterizing isolated strains. Characterizing bacteria of the genus Leptospira

[0003] Bacteria of the genus Leptospira are a direct cause of leptospirosis in humans and animals. Leptospirosis is considered one of the most geographically widespread zoonoses in the world. Currently, infections in humans reach more than 1 million cases registered each year. The clinical picture in humans can vary greatly. Both subclinical infections and severe syndromes with fatal multi-organ disorders may occur. According to recent reports, nearly 60,000 people in the world die every year due to leptospirosis. On the other hand, leptospirosis of farm animals leads to significant economic losses.

[0004] Bacteria of the genus Leptospira are gram-negative bacteria, and thus have a bacterial shield structure typical of this group of microorganisms. It consists of: a cytoplasmic membrane, a peptidoglycan layer in the periplasmic space and an outer membrane surrounding the cell from the outside. The outer membrane is rich in proteins (outer membrane proteins, OMP) and lipopolysaccharide (LPS), which is the main external antigen of these bacteria. LPS of the leptospira has a composition similar to other gram-negative bacteria, but has lower endotoxic activity. Not all bacteria of the genus Leptospira are pathogenic, but it is not possible to morphologically distinguish pathogenic from non-pathogenic strains.

[0005] Initially, leptospira were classified only by serological methods. Live leptospira cell suspensions are agglutinated in the presence of sera from animals and humans infected with these bacteria. As more strains were isolated, antigenic (serological) similarity was shown between some of them. The serological technique has been adopted and refined over the years as a basic tool for routine diagnosis of leptospirosis as well as for describing isolated strains. According to the classical serological classification introduced by Wolf and Broom in 1954, both pathogenic and non-pathogenic leptospira were divided into numerous serovars, determined on the basis of antigenic variation. In addition, these serovars, which give partial cross-reactions with each other in the microscopic agglutination test (MAT), are traditionally grouped into serogroups. Serogroups do not have a taxonomic position but are useful for epidemiological and diagnostic purposes. To date, more than 300 different serovars have been approved, each of which is assigned to one of 23 serogroups. The antigenic diversity observed among leptospira serovars is conditioned by sugar chains of the LPS. LPS has been identified as an immunodominant antigen, both in response to infection and to vaccination. It is also believed to be the only protective antigen identified. Specific antibodies against these antigens protect the host against infection, but only within a specific serovar or at most partially within a specific serogroup. Thus, both natural and postvaccinal immunity are unfortunately directed only against one serovar, they are serovar-specific.

[0006] Leptospira strains adapt to the animal species also at the serovar level. The animal to which a given serovar adapted (evolutionarily adapted) is a natural host for this serovar. Non-natural host animals, including humans, are referred to as accidental hosts. The course of infections in natural and accidental hosts varies. Natural hosts are characterized by high susceptibility to infection, ease of transmission of infection from one animal to another and at the same time very long renal carriage. Although infections in these animals are usually asymptomatic, they can lead to significant economic losses. An example of such adaptation of a serovar to the selected host is the Hardjo serovar in cattle and sheep, Pomona in pigs, Bratislava in pigs and horses or Canicola in dogs. Unlike natural hosts, accidental (incidental) hosts are characterized by low susceptibility to infection, short renal carriage and, above all, inefficient transmission of infections within a given animal species. Knowing whether an animal is infected with an adopted serovar or is accidentally infected by a non-adopted serovar, and thus does not cause long infections spreading in the herd, is important from the point of view of choosing, among others, economically advantageous methods of controlling and combating these infections. Therefore, isolation and full serovar identification of strains occurring in the herd is crucial and is a major challenge for veterinary diagnostics, epidemiological diagnosis and, consequently, allows to optimize the costs of necessary preventive and therapeutic measures.

[0007] In the routine diagnosis of animal leptospirosis, serological tests are used based on detection of the presence of antibodies directed against leptospira using MAT. MAT is based on the reaction of an isolated strain with immune sera obtained from rabbits. The use of MATS in the diagnosis of leptospira indicates only the serogroup causing the infection, but it does not allow to determine the serovar. This test is therefore serogroup-specific, so it allows to assess the affiliation to only one of the 23 known leptospira serogroups.

[0008] Unfortunately, in one serogroup serovars that are the cause of both natural and accidental infections may be present. An example are infections occurring in cattle and sheep. These animals can be natural hosts for the Hardjo serovar classified in the Sejroe serogroup, but they can also be accidental hosts for other serovars in this serogroup, namely the Sejroe and Saxkoebing serovars. Due to the different course of natural and accidental infections, the medicinal -veterinary procedure will be different in the case of the Hardjo serovar (e.g. isolation of infected animals, antibiotic elimination of kidney carriers, vaccination or control of animals newly introduced to the herd) and different in the case of Sejroe or Saxkoebing (elimination of rodents, providing biosecurity to the herd, etc.). Thus, only the identification of the Leptospira serovar can answer the question crucial for further proceedings of whether the diagnosed infection is caused by a serovar adopted for a given animal species, and thus may cause serious losses in breeding, or by a random, transient, non-transferable between animals in the herd. Currently, two methods based on serological reactions are used to assess the serovar affiliation of leptospira field isolates. Both of them have limitations that are difficult to overcome.

[0009] The first known method is the cross-agglutinin absorption test (CAAT) - it identifies the strain to the serovar level. However, conducting it requires (i) live reference strains for each serovar, which are extremely expensive and labor-intensive to maintain in the laboratory; and (ii) control sera from hyperimmunized rabbits against the assessed strain and sera against reference strains of known serovars. For this reason, in practice, this test is performed only by a few reference laboratories in the world, having a rich collection of strains and control sera. CAAT is based on microscopic agglutination of the isolated strain and reference strains with control sera. This reaction is carried out after prior absorption of the antibodies of each serum with a large amount of the heterologous antigen to which it is compared. Based on the results of this test, it is assumed that two strains belong to the same serovar if, after such absorption, less than 10% of the homologous titer remains in each of the two control sera. The more serovars in the serogroup, the more such cross-comparisons of the new strain should be made, which makes the method very labor-intensive and expensive. Although, as a rule, reference laboratories have control sera in their collection against reference strains for individual serovars, sera should also be obtained against the identified strain before testing. Therefore, this method is also timeconsuming, as the immunization of rabbits generally takes 6-10 weeks to obtain adequate antibody titers.

[0010] The second known method is the identification of the serovar using monoclonal antibodies - the use of a microscopic agglutination reaction with the use of monoclonal antibodies significantly accelerates the identification of the serovar. However, due to the weak immunogenic properties of LPS, obtaining such antibodies is not easy. Currently, such antibodies are only available for a few serovars produced by the WHO reference laboratory for leptospirosis in Amsterdam. In the case of other serovars, after repeated attempts to obtain such antibodies, they are still unavailable. The purchase cost for Polish laboratories is relatively high. Genetic methods have introduced a major revolution in the classification of bacteria and they are now the basis of taxonomic classification in bacteriology and allow to understand how bacteria evolved from original and recent ancestors. The basic taxonomic unit, which is defined in genetic terms, is the species. Currently, 68 species have been identified in the genus Leptospira.

[0011] The two above-described divisions used in the leptospira taxonomy, serological and genetic, and thus into serovars and species, do not correlate with each other. Within one species, we have many serovars, but also strains belonging to one serovar can be genetically typed as different species. An example are the strains of the Hardjo serovar, among which there are strains belonging to the Leptospira borgpetersenii species, as well as Leptospira interrogans. This not always consistent duality of identification is one of the biggest technical, economic and scientific problem in the diagnosis of leptospira.

[0012] Although modern taxonomy, including bacteria, is mainly based on species division, in the case of strains belonging to the genus Leptospira, serological identification is still of great practical importance both in scientific research (understanding the mechanisms of development of leptospira infections, adaptation of leptospira to specific animal hosts) and in vaccine prophylaxis and diagnostics of leptospirosis.

[0013] Currently, full typing of Leptospira strains requires a combination of genetic and serological methods to determine the species and serovar. Serological evaluation up to serovar level with CAAT is very labor-intensive, cost-intensive, prolonged in time, and requires procedures using laboratory animals. In order to perform identification up to the serovar level with CAAT, it is necessary to produce a specific rabbit serum for the evaluated isolate, as well as for other known serovars within the serogroup, used for cross-comparison. The growing number of strains isolated in different parts of the world and the huge number of identified serovars (over 300) made the quantitative comparison of the paired strains within only one serogroup by means of this test complicated and, due to the need to use laboratory animals, very prolonged in time (obtaining the approval of the ethics committee for animal experiments and long immunization of animals). In the case of a few serovars, serovar-specific monoclonal antibodies were obtained. However, in most of the described serovars, due to the weak immunogenic properties of the LPS alone, they are unavailable and the only method which still remains is CAAT, which, as mentioned, is very time-consuming, cost-consuming and may raise ethical concerns due to the need to infect and kill animals in order to obtain specific serum.

[0014] These methods, and especially the methods of identifying the serovar, have the aforementioned numerous and difficult to overcome limitations. In particular, the methods currently used for this purpose are very labor- and cost-consuming and require the use of immune sera obtained from rabbits after long-term immunization, which is an additional complication of the methods used so far, among others, for ethical reasons (the need to obtain the consents of the ethics committee for animal experiments).

[0015] The aim of the invention is to propose a method that would shorten the time of determining the serovar affiliation of bacterial strains belonging to the genus Leptospira and allow for omitting procedures using live animals.

[0016] Detailed description of the invention

[0017] The essence of the invention is to determine the serovar by comparing the monosaccharide composition profile of the outer LPS chains. The principle of the method is not based on serological tests, as it has been so far, but on biochemical analyses of the composition of antigenic structures. The method developed according to the invention consists of two steps: the first is the isolation of LPS from the bacterial culture of the tested strain; the second is the identification of the monosaccharide composition by high performance liquid chromatography (HPLC).

[0018] The invention solves the following problems:

[0019] - there is no need to breed and immunize animals with bacteria, including those that are pathogenic also to humans;

[0020] - a long-term, costly and often impossible to efficiently perform procedure for obtaining monoclonal antibodies is not carried out, which also results in making the identification of the serovar independent from the WHO reference laboratory for leptospirosis in Amsterdam;

[0021] - there is no need to cultivate bacterial reference strains, which promotes the method of identification of leptospira, making it independent from the few reference laboratories with control sera against reference strains for individual serovars in their collection - there is no such laboratory in Poland.

[0022] Instead of difficult to obtain reference sera and monoclonal antibodies requiring the cultivation and killing of animals, the invention provides a convenient tool for creating a database and an analytical procedure for the rapid identification of the leptospira serovar, possible to perform in any biochemical laboratory with an ultracentrifuge and HPLC.

[0023] The essence of the invention is a method for determining the monosaccharide composition of the LPS isolated from strains of bacteria of the genus Leptospira and using this information to identify the species, serogroup and serovar of bacteria of the genus Leptospira.

[0024] The test method consists in two consecutive processes aimed at: 1) purification of the LPS from the suspension culture of Leptospira bacteria. and

[0025] 2) separation, purification and labelling of monosaccharides forming LPS of Leptospira spp. and

[0026] 3) analysis of the monosaccharide composition and identification of the serogroup and serovar of bacteria.

[0027] In step 1), it is possible to use the known LPS isolation method with performing phase separation and separation of the LPS by ultracentrifugation of the phenolic fraction.

[0028] In step 2), as the method used to break down the polysaccharides from the LPS weight into component monosaccharides, it is possible to use the known hydrolysis method in trifluoroacetic acid (TFA) solution.

[0029] In step 2), after hydrolysis in TFA, the method used to separate sugars from fatty acids is phase separation using chloroform as a solvent for fatty acid residues. Sugars remain in TFA aqueous solution.

[0030] In step 3), the separated monosaccharides are derivatized at the reducing ends with the 3- methyl-l-phenyl-2-pyrazolin-5-one molecule, which allows the detection of monosaccharides using a spectrophotometric detector during the separation of the formulation using high performance liquid chromatography (HPLC). The procedure used in step 3 is a universal method of sugar composition analysis by means of HPLC.

[0031] During the research leading to the invention, it was unexpectedly found that for all the cases of various bacteria of the genus Leptospira analyzed so far, the monosaccharide composition profiles of the LPS obtained in step 3 allow the identification of the serovar, serogroup and species of bacteria of the genus Leptospira.

[0032] The present invention allows the identification of serovars, serogroups and species of bacteria of the genus Leptospira based on the composition of LPS monosaccharides. The technical approach used requires access to a biochemical laboratory equipped with a high-performance liquid chromatograph. It is also necessary to obtain an axenic culture of the tested type of bacteria of the genus Leptospira. However, the proposed solution is the only one that allows the identification of serovars, serogroups and species of bacteria of the genus Leptospira without the use of sera or antibodies. Moreover, the presented method allows to expand the base of sugar profiles for previously unknown serovars, serogroups and species of the genus Leptospira, which is not absolutely possible using the methods used so far, therefore this solution can be particularly useful in diagnostic conditions where the availability of sera and reference strains collections is limited - as is the case in most veterinary diagnostic centers.

[0033] The described method is unambiguous and can be used to test all serovars, serogroups and species of bacteria of the genus Leptospira.

[0034] The object of the invention in embodiments is shown in the drawings and in the tables, in which:

[0035] Fig. 1 The average percentage share of individual LPS monosaccharides isolated from Leptospira borgpetersenii, Hardjo serovar KR39 strain and Leptospira interrogans, Icterohaemorrhagiae serovar KR93 strain, determined by the percentage share of the area of a given signal in the total of all signal fields analyzed. The error bars represent the standard deviation for the three biological replicates; n=3; * is p <0.05 in the Student's t-test.

[0036] Fig. 2 The average percentage share of individual monosaccharides in LPS from L. interrogans, Hardjo serovar N116 and KR40 strains and L. interrogans, Icterohaemorrhagiae serovar KR93 strain, determined by the percentage share of the area of a given signal in the total of all signal fields analyzed. The error bars represent the standard deviation for the three biological replicates; n=3; * is p<0.05, and ** is p<0.01 in the Tukey's test performed after ANOVA.

[0037] Fig. 3 The average percentage share of individual monosaccharides in LPS of L. interrogans, Copenhagen! serovar M20 strain and L. interrogans, Icterohaemorrhagiae serovar KR 93 strain, determined by the percentage share of the area of a given signal in the total of all signal fields analyzed. The error bars represent the standard deviation for the three biological replicates; n=3; ** is p<0.01 in the Student's t-test.

[0038] Fig. 4 The average percentage share of individual monosaccharides in LPS from L. interrogans, Hardjo serovar N116 strain, KR40 strain and Leptospira borgpetersenii, Hardjo serovar 58 strain and KR39 strain, determined by the percentage share of the area of a given signal in the total of all signal fields analyzed. The error bars represent the standard deviation for the three biological replicates; n=3; *** is p<0.001, and ** is p<0.01 in the Student's test performed on sample groups.

[0039] Fig. 5 The average percentage share of individual monosaccharides in LPS from three different serovars, determined by the percentage share of the area of a given signal in the total of all signal fields analyzed. L. interrogans Hebdomadis serovar Hebdomadis and L. borgpetersenii Sejroe serovars Saxkoebing Mus24 and Sejroe M84 are analyzed. The error bars represent the standard deviation for the three biological replicates; n=3; * is p<0.05, and ** is p<0.01 in the Tukey's test performed after ANOVA.

[0040] Fig. 6 Average percentage share of individual monosaccharides in LPS from L. kirschneiL Serogroup: Grippotyphosa, KR100 isolate, determined by the percentage share of the area of a given signal in the total of all signal fields analyzed. The error bars represent the standard deviation for the three biological replicates; n=3.

[0041] Table 1 Percentage share of individual monosaccharides in the tested LPS samples.

[0042] Table 2 The standard deviation values obtained from the analysis of three separate isolates of each LPS.

[0043] Examples

[0044] Example 1.

[0045] Comparison of Hardjo KR39 (Leptospira borgpetersenii) with Icterohaemorrhagiae KR93 (Leptospira interrogans - two different serogroups: Hardjo and Icterohaemorrhagiae, two different species: L. borgpetersenii and L. interrogans

[0046] In the first example, two isolates of bacteria of the genus Leptospira with different serogroup affiliation and belonging to different genetic species were compared in order to test whether the developed method would show unambiguous and statistically significant differences in the monosaccharide composition of the external LPS chain of these bacteria. An example in which we examine samples where a discrepancy in the sugar composition is expected in the idea of the method.

[0047] In this example, the cultures of the analyzed Leptospira strains were first centrifuged (30 min, 10000G) to obtain bacterial precipitate, from which LPS was then purified using previously published techniques based on aqueous-phenol fractionation. The phenolic fraction comprising most of the LPS of the leptospira was then lyophilized for three days to completely remove moisture from the sample. Such a formulation was weighed and dissolved in deionized water to a concentration of 4 mg / mL. By lyophilizing again 250 pL of such a suspension, formulations comprising 1 mg of LPS were obtained, which were subjected to further analysis. LPS-forming monosaccharides were secreted by hydrolysis of 1 mg of the formulation using 500 pL 2M TFA solution, with which the LPS was treated for 2 hours at 120 °C. After hydrolysis, the formulation was centrifuged and 400 pL of chloroform was added to 400 pL of supernatant to separate LPS-forming lipids. The mixture was shaken for 10 minutes and then centrifuged at 1000 g for 1 minute. The upper aqueous fraction, which comprises LPS-forming monosaccharides, was collected and dried in a nitrogen stream until fully evaporated. For HPLC analysis, monosaccharides found in the sludge remaining after drying were derivatized at the reducing ends with the 3-methyl-l-phenyl-2-pyrazolin-5-one molecule, which was added to sugars using a previously published technique

[0048] (https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC3982747 / ). Labeled PMP monosaccharides were identified by HPLC separation on a Synergi 4 pm Fusion-RP 80 A, LC Column 250 x 4.6 mm and a liquid phase flowing at a rate of 1 mL / min and comprising a mixture of 0.1 M phosphate buffer with pH = 6.6 and acetonitrile of HPLC purity (82: 18 v / v). Monosaccharides from the hydrolysate were identified by comparison to standards of purified sugars. A key innovative element was then the analysis of the chromatogram to determine the monosaccharide composition of the LPS by counting the percentage share of each monosaccharide in the hydrolysate. Then, a comparative analysis of the monosaccharide composition of hydrolysates from the tested Leptospira serogroups was performed in order to distinguish and identify them.

[0049] Comparison of chromatograms and average shares of individual monosaccharides (Fig. 1) in LPS samples revealed significant differences in the monosaccharide composition of samples from different Leptospira serogroups. The N-acetyl-D-glucosamine monosacharide, is characteristic for the serogroup Icterohaemorrhagiae and is not detected in the serogroup Hardjo. In addition, the samples differ significantly in the content of D-mannose, L-ramnose, D-glucose and unknown monosaccharide No. 3 (Student's test, * p<0.05).

[0050] Example 2.

[0051] Comparison of Hardjo N116 and KR40 (L. interrogans) with Icterohaemorrhagiae KR93 (L. interrogans) - two different serogroups: Hardjo and Icterohaemorrhagiae, one species: L. interrogans

[0052] According to the information presented in Example 1, the described procedure makes it possible to distinguish two different serogroups. In order to further validate the method, the monosaccharide composition of the LPS isolated from bacterial cultures belonging to two different serogroups (Hardjo and Icterohaemorrhagiae) but to one genetic species: L. interrogans, SN S analyzed. The studies were conducted according to the same methodology as described in Example 1.

[0053] After obtaining the results, the percentage share of individual monosaccharides (Fig. 2) in the tested samples was calculated. The comparison of the obtained component graphs showed that although the tested organisms belong (genetically) to one species, the previously observed differences between the Hardjo and Icterohaemorrhagiae serogroups still persist. In particular, the N-acetyl-D-glucosamine monosaccharide is still present only in samples from the serogroup Icterohaemorrhagiae. Moreover, D-glucuronic acid, D-glucose, D-xylose and unknown monosaccharide No. 3 have a significantly different percentage share in the tested samples (ANOVA, Tukey's test * p<0.05 and ** p<0.01). Thus, the comparison of the percentage share of monosaccharides allows to distinguish all the tested isolates from each other.

[0054] Example 3.

[0055] Comparison of Icterohaemorrhagiae KR93 (L. interrogans) with Copenhagen! M20 (L. interrogans) - the same serogroup: Icterohaemorrhagiae, the same species: L. interrogans, two different serovars: Icterohaemorrhagiae and Copenhagen!

[0056] A key element of our innovation is the usefulness of the described method in the identification of Leptospira serovars. This element of the analysis is the most difficult to perform with other available research methods. In order to check whether our method allows distinguishing between two serovars of Leptospira, we carried out, in accordance with the previously described methodology, a comparison of the monosaccharide composition of the LPS isolated from Icterohaemorrhagiae KR93 and Copenhagen! M20 strains. Both of these strains belong to the Icterohaemorrhagiae serogroup and belong to one genetic species: L. interrogans. Importantly, previous analyses have shown that they belong to two different serovars, the distinction of which requires a complicated, long-term and costly CAAT analysis.

[0057] In order to compare the monosaccharide composition of LPS Icterohaemorrhagiae KR93 and Copenhagen! M20, we calculated the percentage share of individual sugars observed in the HPLC chromatogram (Fig. 3). The analysis was carried out for three biological replicates of the Leptospira culture. The results indicate that the tested samples differ significantly in the composition: D-xylose constitutes a greater share in LPS of Copenhagen! M20 than in Icterohaemorrhagiae KR93 (Student's test, p-value <0.01). This analysis proves that the described method is able to effectively distinguish the LPS composition of two different serovars belonging to one serogroup.

[0058] Example 4. Comparison of Hardjo N116 and KR40 (L. interrogans) with Hardjo 58 and KR39 (L. borgpetersenii) - one serovar: Hardjo, two different species: L. interrogans and L. borgpetersenii

[0059] In order to confirm the usefulness of our method in identifying as many Leptospira isolates as possible, we decided to analyze LPS obtained from Hardjo-Prajitno and Hardjo-Bovis strains. Both of these strains belong to one serogroup and one serovar. They are clinically important bovine pathogens on the European (N116 and KR40 strains) and the American (strains 58 and KR39) continent. Importantly, genetically these organisms belong to two different species: Leptospira interrogans and Leptospira borgpetersenii.

[0060] In order to compare the composition of LPS, the percentage share of monosaccharides in the tested samples was calculated. The results indicate significantly different levels of mannose, rhamnose and xylose in the tested LPS (Fig. 4, Student's test on groups of samples <0.001 for *** and p<0.01 for **). This means that our method makes it possible to identify the tested strains even if they belong to the same serovar. Such a level of identification was not possible with CAAT methods and is only possible with the use of genetic analysis techniques.

[0061] Example 5:

[0062] Comparison of L. interrogans Hebdomadis serovar and L. borgpetersenii Saxkoebing and Sejroe serovars.

[0063] In order to further confirm the usefulness of our method, we conducted an analysis of the LPS composition in three separate isolates belonging to different serovars. The exact affiliation of the analyzed isolates is: L. interrogans Hebdomadis serovar and L. borgpetersenii Saxkoebing and Sejroe serovars.

[0064] In order to compare the composition of LPS, the percentage share of monosaccharides in the tested samples was calculated. The results indicate significantly different levels of mannose, glucuronic acid, xylose, arabinose and unknown monosaccharide No. 3 (ANOVA, Tukey's test * p<0.05 and ** p<0.01 ). Importantly, the analysis makes it possible to distinguish all the tested isolates, which confirms that for each tested Leptospira serovar, we obtain a specific percentage composition of monosaccharides in LPS.

[0065] Example 6:

[0066] Composition of LPS in Leptospira kirschneri. Serogroup: Grippotyphosa, isolate: KR100. In order to further test the usefulness of our method, we conducted an analysis of the LPS composition obtained from an isolate with a different species and serological affiliation than the previously tested strains. In this example, we analyzed LPS from Leptospira kirschneri. Serogroup: Grippotyphosa, isolate: KR100.

[0067] After performing analysis of the LPS composition, we observed that the molecule in the tested isolate comprises additional sugar, which was not observed during analysis of previous formulations. The sugar was identified as D-galacturonic acid. This observation indicates a high probability of the specificity of the LPS composition of individual Leptospira variants difficult to distinguish by other methods.

Claims

Claims1. A method for identifying a strain of bacteria of the genus Leptospira, characterized in that: a) the LPS produced by the analyzed strain of Leptospira bacteria is isolated, b) hydrolysis of polysaccharides comprised in the isolated LPS is carried out, c) the quantitative and qualitative composition of the monosaccharide composition obtained in step b) is characterized, wherein based on the composition of the monosaccharide composition obtained in step c), the analyzed strain of bacteria of the genus Leptospira is identified.

2. The method according to claim 1, characterized in that in step a) the LPS is isolated by conducting a phase separation to phenol, and optionally by ultracentrifugation.

3. The method according to claim 1, characterized in that in step b) hydrolysis is carried out in trifluoroacetic acid.

3. The method according to claim 1, characterized in that in step b) after hydrolysis, the mixture of sugars is separated from fatty acids by conducting phase separation using chloroform as a solvent for fatty acid residues, wherein the mixture of sugars remains in an aqueous solution.

4. The method according to claim 1, characterized in that in step c) derivatization of monosaccharides comprised in the mixture is carried out at the reducing ends with a 3-methyl- l-phenyl-2-pyrazolin-5-one molecule, and then the resulting mixture is separated using HPLC, wherein the amount of individual monosaccharides is measured by spectrophotometric method.

5. The method according to claim 1, characterized in that in step c) the average percentage share of monosaccharides present in the tested monosaccharide composition is calculated, wherein based on the determined composition of the tested monosaccharide composition, the affiliation of the. Leptospira culture, from which the formulation was obtained, to specific species, serovars and serogroups is determined.

6. The method according to claim 1, characterized in that the compositions of monosaccharide compositions present in LPS specified in Table 1 are considered unique for each of the indicated strains of bacteria of the genus Leptospira.

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