Isolates of Leptospira spp. were cultured from water sources at five different sites in central Iowa in the Midwestern United States and characterized by whole-genome sequencing. Isolates were helix-shaped and motile. Genome sequence analyses determined that the isolates could be clearly distinguished from other species described in the genus Leptospira and included one species that belonged to the pathogen subclade P1, one species that belonged to the pathogen subclade P2 and three species that belonged to the saprophyte subclade S1. The names Leptospira gorisiae sp. nov. (type strain WS92.C1^T=NVSL-WS92.C1^T=KIT0303^T), Leptospira cinconiae sp. nov. (type strain WS58.C1^T=NVSL-WS58.C1^T=KIT0304^T), Leptospira mgodei sp. nov. (type strain WS4.C2^T=NVSL.WS4.C2^T=KIT0305^T), Leptospira iowaensis sp. nov. (type strain WS39.C2^T=NVSL-WS39.C2^T=KIT0306^T) and Leptospira milleri sp. nov. (type strain WS60.C2^T=NVSL-WS60.C2^T=KIT0307^T) are proposed.

Bovine leptospirosis can result in infertility, abortion, placentitis, weak offspring, stillbirths, and decreased milk production. The leading cause of bovine leptospirosis globally is Leptospira borgpetersenii serovar Hardjo. Asymptomatic cattle with leptospirosis serve as reservoir hosts of infection that can shed infectious leptospires for months to years. Sites of bacterial colonization include the kidney or the genital tract, or both, in both cows and bulls. In this observational study, we investigated leptospirosis in a herd of asymptomatic bulls; 24.1% (14/58) were shedding leptospires via urine as confirmed by lipL32 real-time PCR (rtPCR). The viability of leptospires in selected rtPCR-positive bull urine samples was confirmed by culture. Given that bovine semen may also act as a vector of disease transmission, we investigated frozen semen samples for evidence of leptospires; 1.5% (4/268) were lipL32 rtPCR-positive, and, of these, one was culture-positive. All isolates cultured from bull urine or semen were classified as L. borgpetersenii serogroup Sejroe serovar Hardjo. The viability and infectivity of the semen isolate were confirmed in the hamster model of leptospirosis. Our results report for the first time the ability to culture serovar Hardjo directly from frozen semen and highlight the potential role for bull urine and semen in the transmission of bovine leptospirosis.

Background: Leptospirosis is a potentially fatal infectious disease. Therefore, annual revaccination of dogs is recommended, but this can lead to diagnostic interference due to vaccine-induced antibodies. This study determined the prevalence of Leptospira spp.-specific antibodies in 97 healthy adult dogs revaccinated with a 4-serovar vaccine (Nobivac^® L4). Methods: Antibodies were measured with a microscopic agglutination test against 12 serovars before (week 0) and 2, 4, 12, 26, and 52 weeks after revaccination. Logistic regression analysis was performed to determine the presence of pre-revaccination antibodies. Mixed-effect logistic regression analyses and chi-squared tests were used to compare differences between antibodies against vaccine serovars and between vaccine and non-vaccine serovars at different time points. Results: Overall, 63/97 dogs (64.9%) had antibodies against vaccine serovars before revaccination. During the study period, antibodies against $\ge$1 vaccine serovars were detected in all 97 dogs (100.0%). The highest likelihood of detectable antibodies was present in weeks 2 and 4, but 71/97 dogs (73.2%) had antibodies persisting 52 weeks after revaccination. Of 97 dogs, 75 dogs (78.4%) even had antibodies against $\ge$1 non-vaccine serovars. Among those, 19/75 (25.0%) had a fourfold titre increase. Conclusions: These findings suggest that high levels of antibody titres against Leptospira spp. occur frequently and cross-reactivity against non-vaccine serovars is likely. The detection of vaccine-induced antibodies can therefore complicate the diagnosis of leptospirosis.

The microscopic agglutination test (MAT) is the diagnostic standard for canine leptospirosis. However, it is a time-consuming process and does not differentiate between infection- and vaccine-induced antibodies. Canine Leptospira spp.-specific antibody point-of-care (POC) tests provide the rapid detection of immunoglobulin M (IgM) and/or G (IgG). IgM POC tests are considered to become negative more rapidly after vaccination, making them more effective at diagnosing leptospirosis in not-recently vaccinated dogs. This study analysed 582 serum samples of 97 healthy dogs using five different POC tests and the MAT before vaccination and 2, 4, 12, 26, and 52 weeks afterwards. Among the POC tests, three detected IgM antibodies, one detected IgG antibodies, and one detected both IgM and IgG. The results were analysed using mixed-effects logistic regression. Before vaccination, only 2/291 IgM tests were positive (0.7%), compared to 45/194 IgG tests (23.2%). All the POC tests became positive after vaccination, but IgM positivity occurred significantly less frequently (59/1746), especially >4 weeks post-vaccination (7/59 positive results), with 94.5–99.6% specificity compared to 41.4–77.8% in IgG tests. These findings support the use of IgM POC tests in vaccinated dogs, while IgG POC tests are more difficult to interpret.