The serological and genetic diversity of the Leptospira interrogans Icterohaemorrhagiae serogroup circulating in the UK.

Introduction: Strains of Leptospira interrogans belonging to two very closely related serovars, Icterohaemorrhagiae and Copenhageni, have been associated with disease in mammalian species and are the most frequently reported agents of human leptospirosis. They are considered the most pathogenic serovars and represent more than half of the leptospires encountered in severe human infections. Material and Methods: Nineteen such isolates from the United Kingdom - human, domestic and wildlife species - were typed using three monoclonal antibodies (F12 C3, F70 C14 and F70 C24) in an attempt to elucidate their epidemiology. They were further examined by restriction endonuclease analysis (REA), multiple-locus variable-number tandem repeat analysis (MLVA) and lic12008 gene sequence analysis. Results: Monoclonal antibody F12 C3, which is highly specific for Icterohaemorrhagiae and Copenhageni, confirmed that all the strains belonged to these two serovars. Sixteen strains were identified as Copenhageni and three as Icterohaemorrhagiae serovar. Only one restriction pattern type was identified, thus confirming that REA is not able to discriminate between the Icterohaemorrhagiae and Copenhageni serovars. Variable-number tandem-repeat analysis found three loci with differences in the repeat number, indicating genetic diversity between British isolates. Sequences of the lic12008 gene showed that all isolates identified as the Icterohaemorrhagiae serotype have a single base insertion, in contrast to the same sequences of the Copenhageni serotype. Conclusion: Copenhageni is the predominant serovar in the Icterohaemorrhagiae serogroup isolated in British Isles. There is a genetic diversity of MLVA patterns of the isolates but no genetic tool used in the study was able to determine serovars.

Serological study of Leptospira interrogans serovar Copenhageni and L. borgpetersenii serovars Tarassovi and Ballum in beef cattle, sheep and deer in New Zealand.

AIMS: To estimate animal-level seroprevalence of Leptospira interrogans serovar Copenhageni and L. borgpetersenii serovars Ballum and Tarassovi, in beef cattle, sheep and deer on New Zealand farms, and herd/flock-level seroprevalence of any serovar when existing same-sera data for serovars Hardjobovis and Pomona were included, and to determine associations between risk factors and animal-level seroprevalence. METHODS: Banked sera from sheep (n = 82), beef (n = 54) and deer (n = 62) herds/flocks (n = 3,878 animals) from seven regions were analysed using the microscopic agglutination test. Titres of ≥48 were designated positive. Herds/flocks were considered positive if either ≥1, ≥2 or ≥3 animals were positive. Existing same-sera data for serovars Hardjobovis and Pomona were included to establish farm-level any-serovar seropositivity. Factors associated with serological status were analysed using generalised estimating equations. RESULTS: Animal-level seroprevalence for serovars Ballum, Copenhageni, and Tarassovi, respectively, was 13.7 (95% CI = 11.7-16.0)%, 12.6 (95% CI = 10.6-14.7)% and 18.0 (95% CI = 15.7-20.5)% for beef cattle, 10.5 (95% CI = 9.0-12.1)%, 16.7 (95% CI = 14.9-18.6)% and 14.0 (95% CI = 12.4-15.8)% for sheep and 6.6 (95% CI = 5.3-8.2)%, 15.5 (95% CI = 13.5-17.7)% and 3.6 (95% CI = 2.7-4.8)% for deer, respectively. Herd/flock-level seroprevalence for Ballum was 86.6, 52.4 and 39.0% for sheep, 85.2, 52.7 and 33.3% for beef cattle and 50.8, 27.9 and 21.3% for deer at definitions ≥1, ≥2 and ≥3 seropositive animals per species, respectively. For Copenhageni, corresponding data were 95.1, 73.2 and 56.1% for sheep, 68.5, 48.2 and 29.6% for beef cattle and 73.8, 57.4 and 41.0% for deer, and for Tarassovi, 80.5, 59.7 and 45.1% for sheep, 83.3, 68.5 and 61.1% for beef cattle, and 42.6, 16.4 and 4.9% for deer. Seropositivity to all serovars was observed from all regions, with some differences in seroprevalence observed between species and regions, but not between islands. Combining with Hardjobovis and Pomona data, herd/flock-level seropositivity for all animal species and all five Leptospira serovars was 100% at definition ≥1 animal positive, and 97.5 and 96.3% for sheep flocks, 87.8 and 97.8% for beef cattle herds, and 89.3 and 75% for deer herds at ≥2 and ≥3 animals positive, respectively. CONCLUSIONS: Seropositivity to serovars Ballum, Copenhageni and Tarassovi is common in sheep, beef cattle and deer New Zealand and most, or all farms have ≥1 livestock species seropositive to ≥1 serovar. CLINICAL RELEVANCE: Serovars Ballum, Tarassovi and Copenhageni should be considered when clinical or subclinical signs of leptospirosis are observed in sheep, beef cattle or deer. Livestock sector workers are potentially at risk of exposure.

A canine vaccine against Leptospira serovars Icterohaemorrhagiae, Canicola and Grippotyphosa provides cross protection against Leptospira serovar Copenhageni.

Efficacy of the Leptospira components of multivalent vaccine DAPPi-L was previously demonstrated against virulent challenge with three serovars of Leptospira interrogans (Canicola, Icterohaemorrhagiae and Grippotyphosa) carried out 14 days after primary vaccination. In this study we demonstrate that this vaccine provides, two weeks after vaccination, an additional protection (prevention of mortality, clinical signs, renal infection, bacterial excretion, renal carriage and renal lesions) against fatal leptospirosis due to Leptospira interrogans serovar Copenhageni (serovar of major medical importance).

Genomic Comparison Among Global Isolates of L. interrogans Serovars Copenhageni and Icterohaemorrhagiae Identified Natural Genetic Variation Caused by an Indel.

Leptospirosis is a worldwide zoonosis, responsible for more than 1 million cases and 60,000 deaths every year. Among the 13 pathogenic species of the genus Leptospira, serovars belonging to L. interrogans serogroup Icterohaemorrhagiae are considered to be the most virulent strains, and responsible for majority of the reported severe cases. Serovars Copenhageni and Icterohaemorrhagiae are major representatives of this serogroup and despite their public health relevance, little is known regarding the genetic differences between these two serovars. In this study, we analyzed the genome sequences of 67 isolates belonging to L. interrogans serovars Copenhageni and Icterohaemorrhagiae to investigate the influence of spatial and temporal variations on DNA sequence diversity. Out of the 1072 SNPs identified, 276 were in non-coding regions and 796 in coding regions. Indel analyses identified 258 indels, out of which 191 were found in coding regions and 67 in non-coding regions. Our phylogenetic analyses based on SNP dataset revealed that both serovars are closely related but showed distinct spatial clustering. However, likelihood ratio test of the indel data statistically confirmed the presence of a frameshift mutation within a homopolymeric tract of lic12008 gene (related to LPS biosynthesis) in all the L. interrogans serovar Icterohaemorrhagiae strains but not in the Copenhageni strains. Therefore, this internal indel identified can genetically distinguish L. interrogans serovar Copenhageni from serovar Icterohaemorrhagiae with high discriminatory power. To our knowledge, this is the first study to identify global sequence variations (SNPs and Indels) in L. interrogans serovars Copenhageni and Icterohaemorrhagiae.

Passive surveillance of Leptospira infection in swine in Germany.

BACKGROUND: As no current data are available on the prevalence of leptospiral infection in swine in Germany, we analysed laboratory data from diagnostic examinations carried out on samples from swine all over Germany from January 2011 to September 2016. A total of 29,829 swine sera were tested by microscopic agglutination test (MAT) for antibodies against strains of eleven Leptospira serovars. RESULTS: Overall, 20.2% (6025) of the total sample collection tested positive for leptospiral infection. Seropositivity ranged between 16.3% (964) in 2011 and 30.9% (941) in 2016 (January to September only). Of all samples, 11.6% (57.3% of the positives) reacted with only one Leptospira serovar, and only 8.6% (42.7% of the positives) reacted simultaneously with two or more serovars. The most frequently detected serovar was Bratislava, which was found in 11.6% (3448) of all samples, followed by the serovars Australis in 7.3% (2185), Icterohaemorrhagiae in 4.0% (1191), Copenhageni in 4.0% (1182), Autumnalis in 3.7% (1054), Canicola in 2.0% (585), and Pomona in 1.2% (368). Modelling shows that both the year and the reason for testing at the laboratory had statistically strong effects on the test results; however, no interactions were determined between those factors. The results support the suggestion that the seropositivities found may be considered to indicate the state of leptospiral infections in the German swine population. CONCLUSION: Although data from passive surveillance are prone to selection bias, stratified analysis by initial reason for examination and analyses by model approaches may correct for biases. A prevalence of about 20% for a leptospiral infection is most probable for sows with reproductive problems in Germany, with an increasing trend. Swine in Germany are probably a reservoir host for serovar Bratislava, but in contrast to other studies not for Pomona and Tarassovi.

Molecular modeling and in-silico engineering of Cardamom mosaic virus coat protein for the presentation of immunogenic epitopes of Leptospira LipL32.

Expression of Cardamom mosaic virus (CdMV) coat protein (CP) in E. coli forms virus-like particles. In this study, the structure of CdMV CP was predicted and used as a platform to display epitopes of the most abundant surface-associated protein, LipL32 of Leptospira at C, N, and both the termini of CdMV CP. In silico, we have mapped sequential and conformational B-cell epitopes from the crystal structure of LipL32 of Leptospira interrogans serovar Copenhageni str. Fiocruz L1-130 using IEDB Elipro, ABCpred, BCPRED, and VaxiJen servers. Our results show that the epitopes displayed at the N-terminus of CdMV CP are promising vaccine candidates as compared to those displayed at the C-terminus or at both the termini. LipL32 epitopes, EP2, EP3, EP4, and EP6 are found to be promising B-cell epitopes for vaccine development. Based on the type of amino acids, length, surface accessibility, and docking energy with CdMV CP model, the order of antigenicity of the LipL32 epitopes was found to be EP4 > EP3 > EP2 > EP6.

Molecular characterization, serotyping, and antibiotic susceptibility profile of Leptospira interrogans serovar Copenhageni isolates from Brazil.

Leptospira interrogans serogroup Icterohaemorrhagiae is the major serogroup infecting humans worldwide, and rodents and dogs are the most significant transmission sources in urban environments. Knowledge of the prevalent serovars and their maintenance hosts is essential to understand the epidemiology of leptospirosis. In this study, 20 Leptospira isolates were evaluated by pulsed-field gel electrophoresis (PFGE), variable number tandem-repeat analysis (VNTR), serotyping, and determination of antimicrobial resistance profile. Isolates, originated from bovine, canine, human, and rodent sources, were characterized by microscopic agglutination test with polyclonal and monoclonal antibodies and were identified as L. interrogans serogroup Icterohaemorrhagiae serovar Copenhageni. MICs of antimicrobials often used in veterinary medicine were determined by broth microdilution test. Most of tested antibiotics were effective against isolates, including penicillin, ampicillin, and ceftiofur. Higher MIC variability was observed for fluoroquinolones and neomycin; all isolates were resistant to trimethoprim/sulfamethoxazole and sulphadimethoxine. Isolates were genotyped by PFGE and VNTR; both techniques were unable to discriminate between serovars Copenhageni and Icterohaemorrhagiae, as expected. PFGE clustered all isolates in 1 pulsotype, indicating that these serovars can be transmitted between species and that bovine, rodent, and dogs can maintain them in the environment endangering the human population.

Failure of LIC13435 Protein of Leptospira interrogans SerovarCopenhageni to Confer Protection in Immunized Hamsters

Leptospirosis is a re-emergent zoonosis characterized by an acute febrile and systemic illness in humans caused by pathogenic spirochetes belonging to the genus Lepstospira. This disease has global distribution, and it is more frequent in tropical and subtropical areas. The complete genomic sequence of Leptospira species offered the possibility to identify potential vaccine candidates for leptospirosis, since environmental control measures are difficult to implement and there is not an ideal vaccine available for human use. Secreted and surface exposed molecules are potential targets for inducing protective immune response in the host. Although we selected six predicted sequences coding for putative outer membrane proteins with unknown function to be analyzed as vaccinal candidates against leptospirosis and for biological characterization, only the lic13435 gene was expressed and purified. The lic13435 gene is specific for pathogenic leptospires suggesting a possible virulence and/or pathogenicity associated function. The recombinant protein was purified and tested as vaccine candidate against leptospirosis. The immunization with the recombinant protein was able to produce a significant immune response in hamsters. Nevertheless, the animals were not protected against leptospirosis.

Expression and characterization of HlyX hemolysin from Leptospira interrogans serovar Copenhageni Potentiation of hemolytic activity by LipL32

The HlyX, a putative hemolysin identified from the Leptospira genomes, was cloned, expressed in Escherichia coli, purified, and its hemolytic activity was confirmed. Mouse polyclonal antiserum against the recombinant HlyX recognized HlyX-related antigens in a panel of Leptospira species extracts and it was also able to abolish the hemolytic activity of HlyX. A mixture of HlyX and LipL32, a known hemolysin from Leptospira, induced hemolysis in a synergistic way that was fully inhibited by antiserum against either protein. Moreover, sera from patients with leptospirosis also recognized the recombinant HlyX, showing that it is presented to the host immune system during Leptospira infection.