Leptospirosis: classification, epidemiology, and methods of detection. A review

INTRODUCTION

Infectious diseases remain one of the leading causes of high annual morbidity and mortality. Infectious diseases transmitted through animals or vectors (carriers) are called zoonoses. A zoonosis is associated with a specific pathogen that is transmitted from an animal to a human and involves the interaction of humans, animals, and the environment. A meta-analysis from 1940 to the early 21st century showed that 60.3% of new infectious diseases were caused by a rapid spread of zoonotic pathogens (Samrot et al., 2021). Recent outbreaks of SARS and avian influenza demonstrated again the high potential of microorganisms from an animal reservoir to adapt to the human host. Various types of animals, both domestic and wild, are reservoirs of zoonotic pathogens. Considering the high diversity of animal species and the complex life cycles and transmission of pathogens, effective surveillance, prevention, and control of zoonotic diseases pose a public health challenge (Thompson, Kutz, 2019). Echinococcosis, tularaemia, leptospirosis, avian influenza, Hantavirus infection, rabies, and pox viral infections are examples of zoonoses that are transmitted by direct contact with wildlife. These zoonoses are endemic to Europe and occur more often or can emerge again (Rahman et al., 2020). Zoonotic pathogens can circulate in different ecosystems.

Bacteria of the genus Leptospira can cause widespread and potentially lethal bacterial zoonosis called leptospirosis. Considered a neglected tropical zoonotic disease, leptospirosis was recognized as a global public health problem due to its increasing prevalence in both developing and developed countries (Vijayachari et al., 2008). It usually affects vulnerable segments of the population, such as rural farmers and urban slum dwellers. Leptospirosis-induced morbidity and mortality rates are the highest in the world’s poorest regions and the areas where medical care is not regularly carried out (Costa et al., 2015). Leptospirosis is endemic mainly in tropical regions and most often spreads after flooding or heavy rain.

Various wild and domestic mammals can act as reservoir hosts for Leptospira species. An increasing number of studies demonstrate detection of Leptospira spp. in Carnivora, Didelphimorphia, Rodentia, Cingulata, Cetacea, Chiroptera, Afrosoricida, and Primate orders, as well as in Reptilia and Amphibia classes (Cilia et al., 2021). Rodents, which are abundant in urban and peridomestic environments, especially rats Rattus norvegicus and R. rattus, are considered the most important known source of Leptospira infection (Haake et al., 2015). Reservoir hosts carry the pathogen in their renal tubules and excrete pathogenic Leptospira in their urine. Humans and animals can be infected with Leptospira spp. through direct or indirect contact with infected animals or contaminated environments such as soil or water.

According to the Annual Epidemiological Report for 2016 of the European Centre for Disease Prevention and Control, 1319 cases of leptospirosis were reported in 26 countries of the European Union in 2016, of which 783 (59%) were confirmed cases.

In Lithuania, leptospirosis was first diagnosed in 1945. Annually, from 14 to 40 cases were registered between 1995 and 2005 and from three to 20 cases between 2012 and 2016 (ECDC, 2021).

This review is focused on the current knowledge on leptospiral infection, its classification, epidemiology, and detection methods. We are also reviewing the available data on the study of Leptospira in Lithuania in the literature.

TAXONOMY AND CLASSIFICATION

Leptospires belong to the order Spirochaetales, family Leptospiraceae, genus Leptospira. The bacteria belonging to the genus Leptospira are Gram-negative, aerobic, slow-growing, thin, flexible, and tightly coiled spirochetes. Leptospires are usually 0.1 μm wide and 6 to 20 μm long. The cells have pointed ends bent into a typical hook-like shape that allows leptospires to be clearly differentiated from other spirochaetes (Levett, 2001; Samrot et al., 2021). Leptospira spp. consists of an outer membrane with various functional proteins.

The identification of Leptospira isolates has been traditionally based on serological methods, and species identification was determined by the pathogenicity of the isolate. The taxonomy of Leptospira is quite complex due to the high serological diversity. Individual species of Leptospira are divided into serological groups, which are further divided into serovars (Samrot et al., 2021). Initially, after the first description by Stimson (1907), Leptospira were classified into two species, Leptospira interrogans and Leptospira biflexa, which clearly distinguished between pathogenic and saprophytic (non-pathogenic) strains, respectively. These species were differentiated by their nutritional requirements and other phenotypic characteristics. They were further subdivided into specific serovars based on the presence of homologous antigens, defined by the structural heterogeneity of their lipopolysaccharide carbohydrate component (FTA) (approximately 60 L. biflexa serovars and at least 225 L. interrogans serovars) (Levett, 2001). A genotypic classification replaced the phenotypic classification of Leptospira, and there was a move from phenotype-based typing methods towards genotype-based methods such as pulsed field gel electrophoresis (PFGE) and PCR-based methods (Cerqueira, Picardeau, 2009). As 16S rRNA gene sequencing became a common technique for identifying unknown bacterial isolates, it was also applied for the identification of Leptospira species. The phylogeny of Leptospira species is based on a comparative 16S rRNA sequence analysis. Currently, 22 species are distinguished in the genus Leptospira, which are divided into three main groups (clades): pathogenic, intermediate, and saprophytic, and more than 300 serovars. Recently, 12 new species of Leptospira have been discovered in the soil or water, which indicates insufficient knowledge of the biological diversity of this genus (Bulach, Adler, 2018). A genotypic classification complicated the identification of leptospires: some species contain both pathogenic and nonpathogenic serovars, and several serovars are found in more than one species. Thus, neither the serogroup nor serovars can reliably predict Leptospira species, and the identification of the serovar is no longer sufficient to assign an isolate to its correct species (Morey et al., 2006). In addition, more recent studies have demonstrated the genetic heterogeneity of the serovars. The reclassification of Leptospira according to genotypic characters is taxonomically correct and provides a solid basis for further classification of these bacteria (Levett, 2001; Samrot et al., 2021). Other genes have been used to discriminate between species: rpoB, gyrB and ligB (reviewed by Cerqueira, Picardeau, 2009).

METHODS OF LEPTOSPIRA DETECTION

Currently, several different methods are used for the diagnostics of leptospirosis: serological tests (microscopic agglutination test, solid phase assay, enzyme linked immuno-sorbent assay, and indirect haemagglutination assay), direct diagnostic methods (microscopy – phase contrast or dark field microscopy; histochemical staining and immunostaining), culture methods and molecular techniques. Other advanced techniques, such as flow cytometry, have also been developed. In epidemiological studies, identification by species level is not informative, except for the identification of Leptospira for pathogens and for saprophytes. Since each serovar is usually associated with a specific host, the identification of serovars is necessary for epidemiological studies and the development of appropriate prevention strategies (Cerqueira, Picardeau, 2009).

MOLECULAR DETECTION. PCR-BASED METHODS

Modern technology has dramatically improved laboratory procedures, particularly those for the detection, identification, and typing of epidemiologic strains. Molecular methods for DNA detection, such as PCR, are widely used in the diagnosis of leptospirosis in humans and animals. The main advantage of these methods is high sensitivity and specificity. The molecular methods appear to be very useful for the diagnosis of chronic silent leptospirosis in domestic animals.

Several molecular typing techniques have been used for the characterisation of Leptospira strains: bacterial typing methods based on insertion sequence (IS) elements; ribotyping (determination of the restriction fragment length profiles of digested chromosomal DNA probed with rRNA); restriction endonuclease analysis of total genomic DNA and pulsed-field gel electrophoresis (PFGE); randomly amplified polymorphic DNA (RAPD) and arbitrarily primed PCR (AP-PCR); amplified fragment length polymorphism (AFLP). Later, genome analysis identified numerous repeated sequences and many short repetitive DNA sequences (Variable Number of Tandem Repeats) in Leptospira genomes. These sequences have been used for fingerprinting of Leptospira. Conventional PCR is still the most commonly used method for diagnosing leptospirosis in animals, followed by real-time PCR (qPCR), which allowed faster and more sensitive diagnosis compared to conventional PCR (Levett et al., 2005). qPCR is less sensitive to contamination.

Several pairs of primers were described to detect leptospirosis by PCR, some of which were based on specific gene targets, usually 16S or 23S rRNA genes and repeating elements, while others were made from genomic libraries. The 16S rRNA gene was the first molecular marker applied for Leptospira identification, and it is still used today in many studies, mainly for an initial screening. This marker has a good capacity to discriminate between pathogenic, intermediate, and saprophytic leptospires. However, it is ineffective in distinguishing between Leptospira species within a clade. Other universal genes present in bacteria, such as gryB and secY, and Leptospira surface protein genes lipL21, lipL32, lipL41 and ligB are used in PCR-based genotyping (Guernier et al., 2018). The lipL32 gene is currently the most common target used for Leptospira detection, with 48% of studies using this genetic marker (Di Azevedo, Lilenbaum, 2021). Multilocus sequence typing (MLST), a method based on partial sequences of several housekeeping genes (such as adk, icdA, lipL32, lipL41, rrs, secY, pntA, sucA, pfkB, tpiA, mreA, glmU, and fadD), has also been applied to Leptospira spp.

EPIDEMIOLOGY IN THE EUROPEAN UNION

In the past decade, the highest number of confirmed leptospirosis cases in the EU was in 2019, when it reached 1049 cases (ECDC, 2021). In 2020, 22 EU countries reported 565 confirmed leptospirosis cases. Cyprus, Finland, Iceland, Lithuania, Luxemburg, Malta, and Sweden had no confirmed cases in 2020. The countries with the highest number of confirmed cases were Germany, France, and Portugal. The notification rate was 0.14 cases per 100,000 population in the EU. Four countries – Estonia, Portugal, Slovenia and Ireland – had a notification rate of above 0.5 confirmed cases per 100,000 population. In 2020, leptospirosis caused six deaths , compared with ten deaths reported in 2019; in 2018, the fatality rate reached 18. In 2020, almost 90% of people with confirmed cases were hospitalised. As for the Baltic countries, 24 confirmed cases of human leptospirosis were reported in Estonia, 37 in Lithuania, and 24 in Latvia between 2016 and 2020 (Table).

Table. Cases of human leptospirosis in the European Union, 2016-2020 (ECDC)

LEPTOSPIROSIS IN LITHUANIA

Relatively few comprehensive studies on leptospirosis have been reported in Lithuania. Leptospirosis was studied in some administrative districts of Lithuania in 1972–1973. Eight species of rodents and one insectivore species were confirmed to be involved in the epizootic cycle of this disease (Burakauskas, Danilevičius, 1985; Motejunas et al., 1974). The effectiveness of treatment and prevention measures against leptospirosis in cattle and pigs were investigated by researchers at the Veterinary Institute of the Lithuanian Veterinary Academy from 1986 to 1990.

In several regions of the country, different Leptospira serogroups have been isolated from the internal organs and urine of wild (small rodents, wild boars) and domestic (cattle, pigs, horses) animals (Šiugždinienė et al., 2007; Buitkuvienė et al., 2012; Stankevichienė et al., 2016; Jeske et al., 2022).

Šiugždinienė et al. (2007) investigated the prevalence of Leptospira serovars among cattle in Lithuania. The microscopic agglutination test (MAT) was used for the diagnosis of Leptospira serovars. The results of the study showed that the antibodies against L. grippotyphosa (32.46%), L. hebdomadis (25.42%) ser. Kabura, and L. sejroe (18.98%) ser. Poland were the most prevalent in cattle blood sera. The highest serological diversity of different Leptospira serogroups was observed in the Central and Northern counties of Lithuania.

Stankevičienė et al. (2013) investigated the prevalence of leptospirosis in Lithuanian swine farms in 2010. Blood serum samples were randomly collected from 1266 pigs in 28 swine farms in 19 districts. The samples were tested by the microscopic agglutination method. Scientists detected 542 positive reactions to Leptospira in pigs sampled from 17 swine farms. The serovar L. bratislava was the most common in blood samples. Other detected serovars were L. pomona, L. copenhageni, and L. tarassovi.

Four-hundred-and-forty blood serum samples were tested to determine the spread of leptospirosis amongst equine family animals (horses, Shetland ponies, donkeys) in six Lithuanian counties from 2011 to 2015 (Stankevičienė et al., 2016). This study was carried out using a standard serological method, the microscopic agglutination test. Of the tested horses, 18.63% were found serologically positive for leptospirosis. Most of the positive samples (31.8%) were found among horses in Panevėžys County, which was followed by Utena County (19.7%), and Vilnius and Kaunas counties, with about 16.0% in each. Antibodies against serogroups of L. canicola (33.0%), L. copenhagen (26.1%), and L. grippotyphosa (20.9%) were identified in the samples analysed.

Seroprevalence of Leptospira antibodies was also investigated in wild boars. Blood sera samples were collected from 659 healthy wild boars from 42 locations throughout Lithuania during the autumn-winter hunting seasons between 2008 and 2010. The sera of wild boars were analysed using the microscopic agglutination (MA) test. From the examined wild boar sera, 4.6% were positive for different serovars of Leptospira spp. Most frequent were the findings of the antibodies to L. bratislava (3.1%) and L. grippotyphosa (2.1%). Antibodies to L. pomana, L. copenhageni, mixed L. copenhageni – L. bratislava serovars, and L. sejroe were also detected.

A recent study reported the cocirculation of Leptospira spp. and multiple orthohantaviruses in rodents in Lithuania (Jeske et al., 2022). Captured in 23 sites in the country, 1617 rodents and insectivores were screened for zoonotic (re-)emerging Leptospira and orthohantaviruses. Molecular detection methods were used for Leptospira spp. identification. A real-time polymerase chain reaction targeting the lipL32 gene (lipl32-qPCR) was performed for the initial screening of bacteria. Genomospecies identification was done by secY-PCR and multiple locus sequence typing (MLST). Leptospira DNA was found in six rodent species (the striped field mouse Apodemus agrarius, the yellow-necked mouse Apodemus flavicollis, the bank vole Myodes glareolus, the common vole Microtus arvalis, the field vole Microtus agrestis, and the root vole Microtus oeconomus) with an overall mean prevalence of 4.4%. L. kirschneri was identified in the yellow-necked mouse, the striped field mouse, the common vole, and the bank vole. The authors concluded that the detection of re-emerging human pathogenic Leptospira and orthohantaviruses in rodent reservoirs in Lithuania requires increased awareness of public health institutions (Jeske et al., 2022).

CONCLUSIONS

This review summarises the taxonomy, classification, and epidemiology of leptospirosis. The classification of leptospirosis changed over the years, with the genotypic classification replacing the phenotypic classification. Furthermore, as we can see from epidemiology reports, leptospirosis cases in the EU fluctuate from year to year, and the infection still affects hundreds of people across Europe. The review of the studies conducted in Lithuania leads to the conclusion that wild animals such as small rodents and wild boars are natural reservoirs of leptospirosis in particular regions of Lithuania and represent a significant potential source of leptospirosis for other wild and domestic animals as well as for humans. Thus,further studies on leptospirosis and the improvement of molecular diagnostics for pathogen identification are necessary to better understand it and, possibly, even find a way to reduce the numbers of cases and fatalities.

References

Ernestas Urbanskas, Birutė Karvelienė, Jana Radzijevskaja

LEPTOSPIROZĖ: KLASIFIKACIJA, EPIDEMIOLOGIJA IR APTIKIMO METODAI