Leptospirosis is the most neglected widespread zoonosis worldwide. In Spain, leptospirosis reports in people and animals have increased lately. Cats can become infected with Leptospira, as well as be chronic carriers. The aim of this study was to determine serological antibody prevalence against Leptospira sp., blood DNA, and shedding of DNA from pathogenic Leptospira species in the urine of cats in Spain. Microagglutination tests (MAT) and blood and urine TaqMan real-time polymerase chain reaction (PCR) were performed. Leptospira antibodies were detected in 10/244 cats; with 4.1% positive results (95% confidence interval (CI): 2.1–7.18%). Titers ranged from 1:20 to 1:320 (serovars Ballum; Bataviae; Bratislava; Cynopteri; Grippotyphosa Mandemakers; Grippotyphosa Moskva; Pomona; and Proechimys). The most common serovar was Cynopteri. Blood samples from 1/89 cats amplified for Leptospira DNA (1.12%; 95% CI: 0.05–5.41%). Urine samples from 4/232 cats amplified for Leptospira DNA (1.72%; 95% CI: 0.55–4.10%). In conclusion free-roaming cats in Spain can shed pathogenic Leptospira DNA in their urine and may be a source of human infection. Serovars not previously described in cats in Spain were detected; suggesting the presence of at least 4 different species of pathogenic leptospires in the country (L. borgpetersenii; L. interrogans; L. kirschneri; and L. noguchii).

At least two real-time PCRs for the early diagnosis of leptospirosis have been described, evaluated and validated. However, at least one other report suggested adaptation and modification of primers and probes used in these assays since additional Leptospira species have been described and the primers and probe in use possess a serious mismatch to corresponding target sequence. In this study we developed a real-time PCR for detection of pathogenic Leptospira based on the lipL32 gene. The present method consists of generic primers and probes based on target sequence of 10 pathogenic Leptospira species including Leptospira interrogans. The hybridization, annealing and extension temperature (60°C) were optimized as the optimal temperature of the DNA polymerase enzyme which is used in the amplification reaction. The present assay has a high analytical sensitivity and specificity; the calculated diagnostic sensitivity and specificity were 93.0% and 98.3% respectively. Moreover, the present method includes an internal control which enables easy detection of false negative results and an optional extraction control which enables the estimation of the DNA extraction efficiency.

Leptospirosis is a worldwide zoonotic disease, but feline leptospirosis is rarely reported. This study aimed at investigating Leptospira spp. prevalence in cats from southern Italy, evaluating risk factors, clinical findings and laboratory data associated with infection. The serum of 112 cats was investigated by microscopic agglutination test (MAT), detecting anti-Leptospira antibodies against 14 pathogenic serovars. Blood and urine samples were tested by a real-time polymerase chain reaction targeting the lipL32 gene of pathogenic Leptospira. Antibodies against serovars Poi, Bratislava, Arborea, Ballum, Pomona and Lora were detected in 15.3% (17/111) of cats (titers range: 20–320). Leptospira spp. DNA was found in 3% (4/109) of blood and 9% (10/111) of urine samples. The spring season was the only risk factor for urinary Leptospira DNA shedding. Laboratory abnormalities significantly associated and/or correlated with Leptospira spp. positivity were anemia, monocytosis, neutrophilia, eosinopenia, increased alanine aminotransferase activity, hypoalbuminemia and hyperglobulinemia. In the investigated areas, cats are frequently infected by Leptospira spp. and can represent an additional reservoir or sentinel for a risk of infection. Moreover, some laboratory changes could be compatible with a pathogenic effect of Leptospira spp. in the feline host.

Cattle are susceptible to infection with multiple serovars of pathogenic leptospires, resulting in abortion, stillbirth, premature birth, reproductive failure and milk drop syndrome. Cattle also act as a reservoir host for L. borgpetersenii serovar Hardjo which is excreted from renal tubules via urine into the environment where it persists in suitable moist conditions. Our previous work demonstrated that 7% of urine samples from beef cattle were positive for L. borgpetersenii serovar Hardjo by culture and/or the fluorescent antibody test (FAT). In this study, a real-time PCR (rtPCR) assay was applied to determine the relative performance of rtPCR based detection of L. borgpetersenii serovar Hardjo compared to previously reported culture and FAT techniques. Of 42 bovine urine samples positive for leptospires by culture and/or FAT, 60% (25/42) were positive by rtPCR. Of 22 culture-positive samples, 91% (20/22) were rtPCR-positive. Of 32 FAT-positive samples, 50% (16/32) were rtPCR-positive. For 10 samples that were culture-positive but FAT-negative, 90% (9/10) were rtPCR-positive. For 20 samples that were FAT-positive but culture-negative, 25% (5/20) were rtPCR-positive. Collectively, these results indicate that no single assay is optimal, and the use of more than one assay to detect leptospires in urine from naturally infected cattle is recommended.

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.