Prevalence of Anti-leptospiral IgM and Detection of Pathogenic Leptospira Species DNA in Neonates Presenting With Clinical Sepsis in Southwestern Uganda

BackgroundLeptospirosis is an emerging neglected zoonotic disease that presents with nonspecific signs/symptoms and it can be mistaken for other diseases. Due to limited diagnostic capacity and unawareness, data on human leptospirosis particularly in neonates is scarce in many sub-Saharan countries. It has been underreported hindering preventive and control measures in place. The study aimed at determining prevalence of leptospirosis as a cause of febrile illness in neonates using a commercially available IgM ELISA and a quantitative real-time PCR (qPCR). MethodsThis was a descriptive cross-sectional study that included 103 neonatal sepsis cases whose parents/legal guardians gave informed consent. Data on demographic and clinical characteristics was collected using structured data collection form. EDTA whole blood sample was collected from the neonates by trained study nurses. From the samples, IgM ELISA was done using automated analyzers, DNA extracted and qPCR was performed using primers for LipL32, specific for the pathogenic leptospires. ResultsThe prevalence of anti-leptospiral IgM among the neonates as determined by ELISA was 4.3%, where all of them presented with lethargy and poor feeding. No pathogenic Leptospira species DNA was amplified by qPCR.ConclusionsEvidence of leptospirosis was demonstrated in neonatal sepsis cases in this study. The findings suggest considerations of leptospirosis in the differential diagnosis of neonates with sepsis. More data is needed on the real epidemiology, clinical features and burden of leptospirosis in neonates. There is need to include intermediate pathogenic species of Leptospira in the diagnostic qPCR assays.

A Unique Case of Fatal Coinfection Caused by Leptospira spp. and Hepatozoon canis in a Red Fox Cub (Vulpes vulpes)

Red foxes are the most abundant wild carnivore species in Europe commonly exposed to pathogenic Leptospira and Hepatozoon canis. Despite high seroprevalence, the clinical disease caused by these pathogens in red foxes has never been reported. Herein, we report the first-ever case of a fatal Leptospira spp. and H. canis coinfection in a two-month-old red fox cub with acute haemolytic anaemia, mild bronchopneumonia, intraalveolar haemorrhage, and tubulonephrosis. The presence of pathogenic Leptospira spp. DNA was detected in the kidney and lung tissues of the infected animal. In contrast to our previous knowledge, we believe that such fatal cases due to concomitant infection by Leptospira spp. and H. canis, especially in young animals, may commonly occur in nature. However, further studies are required to identify other factors that possibly contribute to the severity and the pathogenic effect of Leptospira spp. and H. canis infections in red foxes.

Co-evolution of Leptospira and Tenrecidae (Afrotheria) on Madagascar

Leptospirosis is one of the most prevalent bacterial zoonoses in the world. The disease is caused by pathogenic Leptospira that are maintained in the kidney lumen of infected animals, mostly mammals, acting as reservoirs and contaminating the environment via infected urine. The investigation of leptospirosis through a One Health framework has been stimulated by notable genetic diversity of pathogenic Leptospira together with a high infection prevalence in certain animal reservoirs. Extensive sampling and associated microbiological and molecular studies of Madagascar's native mammal fauna have revealed a diversity of pathogenic Leptospira with high levels of host-specificity. Native rodents, tenrecids, and bats shelter a number of distinct lineages and species of Leptospira, some of which have also been detected in acute human cases. Specifically, L. mayottensis, first discovered in humans on Mayotte, an island neighboring Madagascar, was subsequently identified in a few species of Malagasy tenrecids, an endemic family. Distinct L. mayottensis lineages were identified in shrew tenrecs (Microgale cowani and Nesogale dobsoni) on Madagascar, and later in spiny tenrecs (Tenrec ecaudatus) on Mayotte. These findings suggest that L. mayottensis (i) has experienced co-evolutionary processes during the radiation of tenrecids on Madagascar, and (ii) has recently emerged in human populations on Mayotte following the introduction of T. ecaudatus from Madagascar. Hitherto, L. mayottensis has not been detected in spiny tenrecs on Madagascar. In the present study, we broaden the investigation of Malagasy tenrecids and describe the presence of L. mayottensis in Malagasy populations of T. ecaudatus, as well as in M. thomasi. These results confirm the hypothesis that L. mayottensis was introduced to Mayotte, presumably via T. ecaudatus, and provide additional data on the co-evolution of Leptospira and Tenrecidae.

Crosstalk between E-Cadherin/β-Catenin and NF-κB Signaling Pathways: The Regulation of Host-Pathogen Interaction during Leptospirosis

Approximately 1 million cases of leptospirosis, an emerging infectious zoonotic disease, are reported each year. Pathogenic Leptospira species express leucine-rich repeat (LRR) proteins that are rarely expressed in non-pathogenic Leptospira species. The LRR domain-containing protein family is vital for the virulence of pathogenic Leptospira species. In this study, the biological mechanisms of an essential LRR domain protein from pathogenic Leptospira were examined. The effects of Leptospira and recombinant LRR20 (rLRR20) on the expression levels of factors involved in signal transduction were examined using microarray, quantitative real-time polymerase chain reaction, and western blotting. The secreted biomarkers were measured using an enzyme-linked immunosorbent assay. rLRR20 colocalized with E-cadherin on the cell surface and activated the downstream transcription factor β-catenin, which subsequently promoted the expression of MMP7, a kidney injury biomarker. Additionally, MMP7 inhibitors were used to demonstrate that the secreted MMP7 degrades surface E-cadherin. This feedback inhibition mechanism downregulated surface E-cadherin expression and inhibited the colonization of Leptospira. The degradation of surface E-cadherin activated the NF-κB signal transduction pathway. Leptospirosis-associated acute kidney injury is associated with the secretion of NGAL, a downstream upregulated biomarker of the NF-κB signal transduction pathway. A working model was proposed to illustrate the crosstalk between E-cadherin/β-catenin and NF-κB signal transduction pathways during Leptospira infection. Thus, rLRR20 of Leptospira induces kidney injury in host cells and inhibits the adhesion and invasion of Leptospira through the upregulation of MMP7 and NGAL.

The oxidative stress response of pathogenic Leptospira is controlled by two peroxide stress regulators which putatively cooperate in controlling virulence

Pathogenic Leptospira are the causative agents of leptospirosis, the most widespread zoonotic infectious disease. Leptospirosis is a potentially severe and life-threatening emerging disease with highest burden in sub-tropical areas and impoverished populations. Mechanisms allowing pathogenic Leptospira to survive inside a host and induce acute leptospirosis are not fully understood. The ability to resist deadly oxidants produced by the host during infection is pivotal for Leptospira virulence. We have previously shown that genes encoding defenses against oxidants in L. interrogans are repressed by PerRA (encoded by LIMLP_10155), a peroxide stress regulator of the Fur family. In this study, we describe the identification and characterization of another putative PerR-like regulator (LIMLP_05620) in L. interrogans. Protein sequence and phylogenetic analyses indicated that LIMLP_05620 displayed all the canonical PerR amino acid residues and is restricted to pathogenic Leptospira clades. We therefore named this PerR-like regulator PerRB. In L. interrogans, the PerRB regulon is distinct from that of PerRA. While a perRA mutant had a greater tolerance to peroxide, inactivating perRB led to a higher tolerance to superoxide, suggesting that these two regulators have a distinct function in the adaptation of L. interrogans to oxidative stress. The concomitant inactivation of perRA and perRB resulted in a higher tolerance to both peroxide and superoxide and, unlike the single mutants, a double perRAperRB mutant was avirulent. Interestingly, this correlated with major changes in gene and non-coding RNA expression. Notably, several virulence-associated genes (clpB, ligA/B, and lvrAB) were repressed. By obtaining a double mutant in a pathogenic Leptospira strain, our study has uncovered an interplay of two PerRs in the adaptation of Leptospira to oxidative stress with a putative role in virulence and pathogenicity, most likely through the transcriptional control of a complex regulatory network.

Pengaruh Sodium Hipoklorit dan Kalsium Hipoklorit terhadap Daya Hidup Leptospira Patogenik

Leptospirosis is still a health problem in the word. Leptospirosis can be transmitted to humans trough contact with environment infected with pathogenic Leptospira. Efforts to control pathogenic Leptospira in the environment can be done one of them by disinfecting. Effective desinfectants for the control of pathogenic Leptospira include the active ingredients Calcium hypochlorite 60% and Sodium hypochlorite 5,25%. This study aims to determine the effectiveness of Calcium hypochlorite 60% and Sodium hypochlorite 5,25% against the life power of pathogenic Leptospira. This research is a laboratory study with pure experimental design. Serovar of pathogenic Leptospira used is Leptospira icterohaemorrhagiae. Leptospira icterohaemorrhagiae desnsity used as much as 5.7 x 106 Leptospira/ml. The concentrations of desinfectans and bacteria are: 5 ppm, 50 ppm, 500 ppm, 1000 ppm, 2000 ppm, 5000 ppm, 10.000 ppm, and 25.000 ppm. Observations are made every ten minutes and twenty minutes with three repeats. The results showed that Leptospira icterohaemorrhagiae could not survive when tested on 60% calcium hypochlorite and 5.25% sodium hypochlorite, respectively, at a concentration of 2000 ppm and a concentration of 1000 ppm. Statistical results showed there was a significant difference in the concentration of each disinfectant active ingredient. Thus, a disinfectans of chlorine with the active ingredient Calcium hypochlorite 60% and Sodium hypochlorite 5,25% effectively affects the viability of the Leptospira icterohaemorrhagiae. Keywords : Sodium Hypochlorite, Calcium Hypochlorite, Leptospira, Disinfectant Abstrak Leptospirosis masih menjadi masalah kesehatan di dunia. Leptospirosis dapat menular ke manusia lewat kontak dengan lingkungan yang mengandung Leptospira patogenik. Upaya pengendalian Leptospira patogenik di lingkungan dapat dilakukan salah satunya dengan pemberian disinfektan. Disinfektan yang efektif untuk pengendalian Leptospira patogenik diantaranya adalah bahan aktif kalsium hipoklorit 60% dan sodium hipoklorit 5,25%. Penelitian ini bertujuan mengetahui efektivitas bahan aktif kalsium hipoklorit 60% dan sodium hipoklorit 5,25% terhadap daya hidup Leptospira patogenik. Penelitian ini merupakan penelitian skala laboratorium dengan rancangan eksperimental murni. Serovar Leptospira patogenik yang digunakan adalah Leptospira icterohaemorrhagiae. Kepadatan Leptospira icterohaemorrhagiae yang digunakan sebanyak 5,7 x 106 Leptospira/ml. Konsentrasi campuran disinfektan dan bakteri yaitu: 5 ppm, 50 ppm, 500 ppm, 1000 ppm, 2000 ppm, 5000 ppm, 10.000 ppm, dan 25.000 ppm. Pengamatan dilakukan tiap 10 menit dan 20 menit dengan tiga kali ulangan. Hasil penelitian menunjukkan Leptospira icterohaemorrhagiae tidak dapat hidup saat diujikan ke kalsium hipoklorit 60% dan sodium hipoklorit 5,25% berturut-turut pada konsentrasi 2000 ppm dan konsentrasi 1000 ppm. Hasil statistik menunjukkan ada perbedaan signifikan dari besaran konsentrasi masing-masing bahan aktif disinfektan. Dengan demikian, disinfektan klorin dengan bahan aktif calcium hypochlorite 60% dan sodium hypochlorite 5,25% efektif memengaruhi daya hidup Leptospira icterohaemorrhagiae. Kata kunci: Sodium hipoklorit, Kalsium hipoklorit, Leptospira, Disinfektan

Tracking Animal Reservoirs of Pathogenic Leptospira: The Right Test for the Right Claim

Leptospirosis is the most prevalent bacterial zoonosis worldwide and, in this context, has been extensively investigated through the One Health framework. Diagnosis of human leptospirosis includes molecular and serological tools, with the serological Microscopic Agglutination Test (MAT) still being considered as the gold standard. Mammals acting as reservoirs of the pathogen include species or populations that are able to maintain chronic infection and shed the bacteria via their urine into the environment. Animals infected by Leptospira are often identified using the same diagnosis tool as in humans, i.e., serological MAT. However, this tool may lead to misinterpretations as it can signal previous infection but does not provide accurate information regarding the capacity of animals to maintain chronic infection and, hence, participate in the transmission cycle. We employ in this paper previously published data and present original results on introduced and endemic small mammals from Indian Ocean islands to show that MAT should not be used for the identification of Leptospira reservoirs. By contrast, serological data are informative on the level of exposure of animals living in a specific environment. We present a sequential methodology to investigate human leptospirosis in the One Health framework that associates molecular detection in humans and animals, together with MAT of human samples using Leptospira isolates obtained from reservoir animals occurring in the same environment.

Current epidemiological situation on leptospirosis in the territories adjacent to Lake Baikal

Background. In presence the epizootiological situation on leptospirosis in the territories adjacent to Lake Baikal is not studied well. The showing up of natural focus, discovering of host animal’s species composition and etiological structure of leptospirosis causative agents are essential to plan any epidemiological response and to organize the specific prophylaxis.Aim: to analyze contemporary characteristics of leptospirosis natural foci at the territories adjacent to Lake Baikal.Materials and methods. From 2011 till 2020 the territory of nine Irkutsk Region's administrative districts was investigated. Trapping has been done at four kind of sites (wetland, meadow, forest-shrub, steppificated meadow) and boroughs. 1152 small mammals which belonged to 35 species were collected. Collected samples were studied by complex of methods. Serogroup belonging was detected by micro agglutination and decomposition test, and genomic species – by Microflex LT mass analyzer.Results. A leptospirosis natural foci in the Irkutsk city boundary was newly founded and there were isolated (from small mammals) four pathogenic cultures of leptospiras belonging to Javanica serogroup. Main hosts of causative agents at the territories adjacent to Lake Baikal were defined (insect-eating mammals: tundra shrew, largetoothed Siberian shrew, even-toothed shrew, shrew-mouse, and rodents: root vole, ground vole, narrow-skulled vole, sewer rat). By micro agglutination were defined pathogenic leptospirosis serogroups (Icterohaemorrhagiae, Grippotyphosa, Javanica, Pomona, Sejroe, Autumnalis, Australis), all over 2.4 ± 0.45 % seropositive. Antibody dilution at survey animals ranged from 1 : 20 to 1 : 640. On the grounds of PCR results the mean value of infected animals in studied sample was 16.4 ± 1.14 %. Positive PCR findings were at 19 from 35 animal species.Conclusion. At the territories adjacent to Lake Baikal In the Baikal region, the proportion of animals that have had contact with pathogenic leptospira is quite high. Natural focus of this infection are highly pervasive in this region, they are evolved mainly in dewy sites.