Zbigniew Arent

Isolation of Leptospires from genital tract of sheep

Leptospirosis is an economically important infectious disease of domestic livestock. Renal infection is the most common microbiological sequel to infection in most animal species, but in the case of host-maintained infections, such as serovar Hardjo infection in cattle (both type Hardjobovis and Hardjoprajitno) or serovar Bratislava infection in pigs, persistent genital tract infection is a major feature (Ellis and others 1986a, b, c, d). Evidence of significant serological prevalences to serovar Hardjo (Egan and Yearsley 1987, Cerri and others 2003, Herrmann and others 2004, Ridler and others 2005, Anonymous 2012, Martins and other 2012), abattoir studies (Bahaman and others 1980, Dorjee and others 2008), experimental studies and the apparent independence of sheep infections from cattle (Pekelder and others 1993) would indicate that sheep may also act as an alternative maintenance host for serovar Hardjo (Cousins and others 1989, Gerritsen and others 1994, Farina and others 1996). While serovar Hardjo has been recovered …

Altodouro, a new Leptospira serovar of the Pomona serogroup isolated from rodents in northern Portugal

A Leptospira strain (designated RIM 139) was isolated from the kidney of a house-mouse, Mus musculus, in Trás-os-Montes e Alto Douro region of northern Portugal. The isolate showed typical leptospiral motility and morphology under dark-field microscopy and was pathogenic for hamsters. Species determination was carried out on basis of PCR products generated by species-specific primers and by sequencing of the secY gene. Putative serogroup typing was performed using the microscopic agglutination test (MAT) with a panel of rabbit anti-Leptospira sera representative of the major pathogenic serogroups. Serovar identification was carried out by a combination of monoclonal antibodies and cross-agglutinin absorption test (CAAT). The novel nature of the strain was confirmed by restriction endonuclease analysis (REA). Results showed that RIM 139 represents a new serovar. The name Altodouro is proposed for this new serovar. Speciation findings - PCR analysis of the ompL1 gene and sequencing of the secY gene - indicated that it belonged to Leptospira kirschneri as did amplification with G1/G2 and B64-I/B64-II the primer sets.

Leptospira interrogans serovars Bratislava and Muenchen animal infections: Implications for epidemiology and control

Strains of Leptospira interrogans belonging to two very closely related serovars - Bratislava and Muenchen - have been associated with disease in domestic animals, in particular pigs, but also in horses and dogs. Similar strains have also been recovered from various wildlife species. Their epidemiology is poorly understood. Two hundred and forty seven such isolates, from UK domestic animal and wildlife species, were examined by restriction endonuclease analysis in an attempt to elucidate their epidemiology. A representative sub-sample of 65 of these isolates was further examined by multiple-locus variable-number tandem repeat analysis and 22 by secY sequencing. Ten restriction pattern types were identified. The majority of isolates fell into one of three restriction endonuclease analysis pattern types designated B2a, B2b and M2a. B2a was ubiquitous and was isolated from 10 species and represented the majority of the horse and all dog isolates. B2b was very different, being isolated only from pigs, indicating that this type was maintained by pigs. The pattern M2a was reported for the majority of isolates from pigs but also was common in small rodents isolates. Five restriction pattern types were found only in wildlife suggesting that they are unlikely to pose a disease threat to domestic animals. Multiple-locus variable-number tandem repeat analysis identified six clusters. The REA types B2a and B2b were all found in one MLVA cluster while the majority of the M2a strains examined occurred in another cluster. The secY sequencing detected only one sequence type, clustered with other serovars of Leptospira interrogans.

Molecular studies on European equine isolates of Leptospira interrogans serovars Bratislava and Muenchen

Strains of Leptospira interrogans belonging to two very closely related serovars – Bratislava and Muenchen – are known to cause widespread infection of the horse population in many parts of the world. Conventional serological typing of isolates has been unable to differentiate between wildlife, pig, dog and possibly horse maintained isolates and therefore has been unable to provide further insight into their diversity and the relationship between them. Twenty-one such European isolates of serovar Bratislava and Muenchen were examined by restriction endonuclease analysis and multiple-locus variable-number tandem repeat analysis in an attempt to elucidate their epidemiology. The restriction pattern types were identified and fell into one of four REA designed pattern types, B1, B2a, M1, M2a. Nine strains from Northern Ireland and two from Germany belonged to B2a, which is a ubiquitous strain being originally isolated from a large number of wild and domestic animal species in the UK. Five strains were identified as B1 and they came from Portugal, The Netherlands, Germany and Northern Ireland; three strains isolated in Germany belong to M1; two strains belonged to M2a. Genotypes B1 and M1 have, with the exception of one hedgehog isolate, been recovered only from horses and it may indicate their adaptation to this species.

Emerging Infectious Disease Implications of Invasive Mammalian Species: The Greater White-Toothed Shrew (Crocidura russula) Is Associated With a Novel Serovar of Pathogenic Leptospira in Ireland.

The greater white-toothed shrew (Crocidura russula) is an invasive mammalian species that was first recorded in Ireland in 2007. It currently occupies an area of approximately 7,600 km2 on the island. C. russula is normally distributed in Northern Africa and Western Europe, and was previously absent from the British Isles. Whilst invasive species can have dramatic and rapid impacts on faunal and floral communities, they may also be carriers of pathogens facilitating disease transmission in potentially naive populations. Pathogenic leptospires are endemic in Ireland and a significant cause of human and animal disease. From 18 trapped C. russula, 3 isolates of Leptospira were cultured. However, typing of these isolates by standard serological reference methods was negative, and suggested an, as yet, unidentified serovar. Sequence analysis of 16S ribosomal RNA and secY indicated that these novel isolates belong to Leptospira alstonii, a unique pathogenic species of which only 7 isolates have been described to date. Earlier isolations were limited geographically to China, Japan and Malaysia, and this leptospiral species had not previously been cultured from mammals. Restriction enzyme analysis (REA) further confirms the novelty of these strains since no similar patterns were observed with a reference database of leptospires. As with other pathogenic Leptospira species, these isolates contain lipL32 and do not grow in the presence of 8-azagunaine; however no evidence of disease was apparent after experimental infection of hamsters. These isolates are genetically related to L. alstonii but have a novel REA pattern; they represent a new serovar which we designate as serovar Room22. This study demonstrates that invasive mammalian species act as bridge vectors of novel zoonotic pathogens such as Leptospira.

Characterization of the molecular chaperone ClpB from the pathogenic spirochaete Leptospira interrogans

Leptospira interrogans is a spirochaete responsible for leptospirosis in mammals. The molecular mechanisms of the Leptospira virulence remain mostly unknown. Recently, it has been demonstrated that an AAA+ chaperone ClpB (a member of the Hsp100 family) from L. interrogans (ClpBLi) is not only essential for survival of Leptospira under the thermal and oxidative stresses, but also during infection of a host. The aim of this study was to provide further insight into the role of ClpB in the pathogenic spirochaetes and explore its biochemical properties. We found that a non-hydrolysable ATP analogue, ATPγS, but not AMP-PNP induces the formation of ClpBLi hexamers and stabilizes the associated form of the chaperone. ADP also induces structural changes in ClpBLi and promotes its self-assembly, but does not produce full association into the hexamers. We also demonstrated that ClpBLi exhibits a weak ATPase activity that is stimulated by κ-casein and poly-lysine, and may mediate protein disaggregation independently from the DnaK chaperone system. Unexpectedly, the presence of E. coli DnaK/DnaJ/GrpE did not significantly affect the disaggregation activity of ClpBLi and ClpBLi did not substitute for the ClpBEc function in the clpB-null E. coli strain. This result underscores the species-specificity of the ClpB cooperation with the co-chaperones and is most likely due to a loss of interactions between the ClpBLi middle domain and the E. coli DnaK. We also found that ClpBLi interacts more efficiently with the aggregated G6PDH in the presence of ATPγS rather than ATP. Our results indicate that ClpB’s importance during infection might be due to its role as a molecular chaperone involved in reactivation of protein aggregates.

Complete Genome Sequence of Leptospira alstonii Serovar Room22 Strain GWTS #1

ABSTRACT We report here the complete genome sequence of Leptospira alstonii serovar Room22 strain GWTS #1. This is the first isolate of L. alstonii to be cultured from a mammal and in western Europe, and it represents a new serovar of pathogenic leptospires.

Isolation and Identification of Putative Protein Substrates of the AAA+ Molecular Chaperone ClpB from the Pathogenic Spirochaete Leptospira interrogans

Bacterial ClpB is an ATP-dependent Hsp100 chaperone that reactivates aggregated proteins in cooperation with the DnaK chaperone system and promotes survival of bacteria under stress conditions. A large number of publications also indicate that ClpB supports the virulence of bacteria, including a pathogenic spirochaete Leptospira interrogans responsible for leptospirosis in both animals and humans. However, the exact role of ClpB in bacterial pathogenicity remains poorly characterized. It can be assumed that ClpB, due to its role as the molecular chaperone, mediates refolding of essential bacterial proteins, including the known virulence factors, which may become prone to aggregation under infection-induced stresses. In this study, we identified putative substrates of ClpB from L. interrogans (ClpBLi). For this purpose, we used a proteomic approach combining the ClpB-Trap affinity pull-down assays, Liquid chromatography-tandem mass spectrometry (LC-MS-MS/MS), and bioinformatics analyses. Most of the identified proteins were enzymes predominantly associated with major metabolic pathways like the tricarboxylic acid (TCA) cycle, glycolysis–gluconeogenesis and amino acid and fatty acid metabolism. Based on our proteomic study, we suggest that ClpB can support the virulence of L. interrogans by protecting the conformational integrity and catalytic activity of multiple metabolic enzymes, thus maintaining energy homeostasis in pathogen cells.

Leptospira interrogans serogroup Pomona infections in the UK: is there a real threat for farm animals?

Current typing of Leptospira strains requires a combination of both serological and molecular methods. Study of the epidemiology of strains within the Pomona serogroup has proved problematic due to the limitations of serological typing methods. This is particularly true of the two main complexes within this group, namely the Pomona, Monjakov, Kennewicki cluster in Leptospira interrogans species and the Mozdok, Tsaratsovo, Kunming, Altodouro cluster in Leptospira kirschneri. Strains from the south of England have been shown previously to belong to L kirschneri serovar Mozdok, strains of which are maintained by small rodents in western Europe. While they may occasionally cause disease in domestic animals, they are unlikely to be of economic importance. In contrast, L interrogans serovar Pomona type Kennewicki strains have been shown to be significant animal pathogens, especially in north America, while L interrogans serovar Pomona types Pomona and Monjakov have not been associated with significant economic loss. The purpose of this study was to examine 10 UK serovar Pomona isolates to assess type as a means of assessing the possible risk they pose to British livestock. All isolates were identified as L interrogans serovar Pomona type Pomona and were therefore unlikely to pose a significant threat to domestic animals in the UK.

First report of Leptospira infections in red deer, roe deer, and fallow deer in Poland

Abstract Introduction: Recently in Europe an increase in the population of red deer (Cervus elaphus), roe deer (Capreolus capreolus), and fallow deer (Dama dama) has been observed. Research on the prevalence of Leptospira infections in Polish cervids has been performed for the first time. Material and Methods: During 2014/2015 hunting season, 147 blood samples from red deer, roe deer, and fallow deer were collected. The animals originated from different geographical regions across Poland. Serum samples were tested by microscopic agglutination test (MAT) for the presence of specific antibodies to the following Leptospira serovars: Icterohaemorrhagiae, Grippotyphosa, Sejroe, Tarassovi, Pomona, Canicola, Bratislava, Hardjo, Ballum, Zanoni, Hebdomadis, and Poi. Results: Serum antibody titres specific to Grippotyphosa, Pomona, and Zanoni serovars were found; none of the sera were positive for any of the other serovars. Out of 147 serum samples only 7 were positive, which gave an overall prevalence of 4.8% in the tested animal population. Conclusion: The low Leptospira antibody titres along with the low number of positive serum samples in deer indicate that these animals may not act as significant reservoirs of Leptospira for either humans or animals in Poland.