Leptospirosis is an infectious disease caused by the spirochete bacteria Leptospira spp. and is commonly found throughout the world. Diagnosis of leptospirosis performed by culture and microscopic agglutination tests is laborious and time-consuming. Therefore, we aimed to develop a novel immunochromatography (ICG)-based method for detecting Leptospira antigen in the urine of patients and animals. We used the 1H6 monoclonal antibody (MAb), which is specific to the lipopolysaccharide (LPS) that is common among Leptospira spp. The MAb was coupled to 40-nm-diameter colloidal gold, and the amounts of labeled antibody and immobilized antibody were 23 g and 2 g per test, respectively. Several strains of Leptospira and other bacterial species were used to evaluate the sensitivities and specificities of the assays we developed. The detection limit of the assays was 10 6 cells/ml when disrupted whole bacterial cells were used. The assays were Leptospira specific since they did not cross-react with non-Leptospira bacteria used in the study. Application of diagnostic assays was done on the urine samples of 46 Leptospira-infected hamsters, 44 patients with suspected leptospirosis, and 14 healthy individuals. Pretreatment of the urine samples by boiling and centrifugation (for ultrafiltration and concentration) eliminated nonspecific reactions that occurred in the assay. The sensitivity and specificity of the ICG-based lateral flow assay (LFA) were 89% and 87%, respectively, which were higher than those of the dipstick assay, which were 80% and 74%, respectively. In summary, this ICG-based LFA can be used as an alternative diagnostic assay for leptospirosis. Further development is still necessary to improve the assay.

Rats are known to be the most important reservoirs of Leptospira spp. However, the leptospiral dose and age at which rats become resistant to Leptospira infection are not yet well elucidated. Aimed to characterize leptospirosis in rat pups, we found that suckling pups (4-, 7-, and 14-day old) are susceptible to leptospires and resistance starts from the weaning age (23-day old). Susceptibility of rat pups was also affected by the infecting dose of the organisms. Jaundice, decrease in body weight, and neurological symptoms prior to moribundity was evident in infected suckling pups. However, 23-day-old infected pups did not manifest any pathological changes and were able to survive the infection similar to adult rats. Based on these results, we propose the suckling rat pup as a novel animal model of human leptospirosis to investigate pathogenesis, development of host resistance, and the mechanisms involved in rats becoming maintenance hosts for leptospires.

Leptospirosis is a worldwide zoonosis. The importance of urban leptospirosis is recognized in Japan: urban rats carry pathogenic leptospires and people acquire these pathogens through contact with surface water or soil contaminated by the urine of the infected animals. To determine the current Leptospira carriage rate in urban rats, 29 wild rats were trapped in the central area of Fukuoka and strains isolated from their kidneys and urine analyzed. When semi-solid Korthof's medium containing 0.1% agar was used for isolation, 72.2% and 30.8% of the kidney and urine cultures, respectively, were found to be Leptospira-positive. The isolates belonged to Leptospira interrogans, and were classified into two groups (serogroups Pomona and Icterohaemorrhagiae) based on the results of gyrB sequence analysis and microscopic agglutination testing (MAT). Strains belonging to serogroup Icterohemorrhagiae grew well in liquid medium. On the other hand, serogroup Pomona isolates multiplied very little in liquid medium, but did grow in a semi-solid medium. Although strains belonging to serogroup Pomona have not been recognized as native to Japan, this strain may be widely distributed in urban rats. Representative strains from each group were found to be highly pathogenic to hamsters. Our findings should serve as a warning that it is still possible to become infected with leptospires from wild rats living in inner cities of Japan. Furthermore, the use of semisolid medium for culture will improve the isolation rate of leptospires from the kidneys of wild rats.

No abstract