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The early diagnosis of canine leptospirosis is still a challenge for clinicians that in most cases only have basic routine tests as diagnostic tools. This study aimed to isolate and characterize leptospires from the blood of dogs with clinical signs suggestive of leptospirosis and to evaluate the results of some laboratory tests in hamsters experimentally infected with these isolates. The blood of 22 animals was submitted to bacteriological culturing, and the isolates recovered were characterized by serogrouping, partial DNA sequencing (secY), and multiple locus variable-number tandem repeat analysis (MLVA). Golden Syrian hamsters were infected with isolated leptospires and submitted to serology using microscopic agglutination test (MAT), polymerase chain reaction (PCR), complete blood counts, and serum biochemistry. Three isolates (13.63%) were obtained, and all were assigned to the serogroup Icterohaemorrhagiae. The DNA sequencing results showed that all the isolates belonged to the species Leptospira interrogans, and the MLVA results evidenced that the profile of the strains was compatible with Copenhageni/Icterohaemorrhagiae serovars. Regarding laboratory tests performed on hamsters infected with these isolated strains, marked leukocytosis by neutrophilia and monocytosis was observed, associated with an accentuated increase in renal and hepatic indicators. In addition, a titer of 800 was detected by MAT for only the serogroup Icterohaemorrhagiae, and blood, liver, kidneys, and lung samples were PCR positive. The presence of these isolates suggests that the dogs were infected with strains carried by rodents, and significant changes were found in routine laboratory tests, which, when correlated, can help veterinarians with early diagnosis. [ABSTRACT FROM AUTHOR]

• A Leptospira strain was recovered from urine of a water buffalo. • The isolate was assigned to the species L. borgpetersenii and the Sejroe serogroup. • This was the first isolation of a strain other than the subgroup Wolffi in Brazil. • The strain was detected in the reproductive tract of experimentally infected hamsters. The isolation of leptospires from buffaloes worldwide is still limited to a few strains. Thus, the aim of this study was to describe the first Leptospira isolate from buffalo urine, assigned to the Sejroe serogroup, which does not belong to the Wolffi subgroup, traditionally isolated in Brazil. A total of 244 urine samples of water buffaloes (Bubalus bubalis) raised in the Brazilian Amazon were subjected to bacteriological culturing and polymerase chain reaction (PCR) for the detection of leptospires. The obtained isolate was characterized by serogrouping using polyclonal antibodies, partial DNA sequencing, Hardjo-Bovis-specific PCR, multiple-locus variable-number tandem repeat analysis (MLVA/VNTR) and experimental infection in hamsters. PCR was performed on the urine samples; 11/244 were positive (4.5 %) for Leptospira , and only one isolate was recovered (0.4 %). Regarding characterization, the isolate was assigned to the Sejroe serogroup with high titers (12,800) for the Saxkoebing and Sejroe serovar antisera. The isolate was negative for Hardjo-Bovis-specific PCR, and the species Leptospira borgpetersenii was identified by DNA sequencing. The MLVA results showed that the VNTR profile of the isolate was 1−2-5, compatible with that of serovars Sejroe/Istrica. In the experimental infection in hamsters, the animals did not develop clinical signs, and no macroscopic lesions were observed on the organs at necropsy; however, the strain was detected in the kidneys, uterus, and testicles of the animals. The isolate described herein highlights infection by Sejroe strains that may be overlooked in buffaloes and that may be different from those normally isolated and used in serological studies. [ABSTRACT FROM AUTHOR]

This study aimed to develop and evaluate a pooled antigen for use in the macroscopic slide agglutination test (MSAT) to detect cattle positive for the Sejroe serogroup. To this end, 193 bovine serum samples from different Pará State regions (Amazonia) were subjected to a reference microscopic agglutination test (MAT) for the serological diagnosis of leptospirosis using 11 serovars representing the Sejroe serogroup: Hardjo-prajitno; Hardjo-bovis; Sejroe; Wolffi; Guaricura (Bov.G.); Guaricura (M4/98); Ricardi; Gorgas; Recreo; Polonica and Medanensis. The three most prevalent serovars in the MAT were selected for the development of a pooled antigen for use in MSAT; subsequently, the 193 serum were assessed with the macroscopic slide agglutination test (MSAT) containing the developed antigen. The Kappa test was used to determine the general agreement between the MAT and MSAT results. As a result, of the 193 serum samples, 155 (80.3%) were reactive, and 38 (19.7%) were non-reactive in the MAT; Hardjo-prajitno, Wolffi and Medanensis were the three most prevalent serovars. Of the 193 serum samples tested in the MSAT using the developed pooled antigen, 114 were reactive (59.0%), and 79 (41.0%) were non-reactive; the Kappa coefficient was 0.52 (CI 95%, 0.40–0.63), indicating moderate agreement between the two tests. The MSAT with the pooled antigen including the most prevalent serovars detected bovines with the Sejroe serogroup exposure, mainly in animals with high titters in the MAT, and could be used to screen herds suspected of acute infection by this serogroup in Pará State. • The macroscopic slide agglutination test (MSAT) was first used in Brazil to detect cattle exposed to Sejroe serogroup. • The pooled antigen used on MSAT was composed by Hardjo-prajitno, Wolffi and Medanensis serovars. • The addition of Ponceau S dye to pooled antigen favored the visualization of reactive samples. • The MSAT using this developed antigen could be used for initial screening in herds in Amazonia, especially in remote areas. [ABSTRACT FROM AUTHOR]

The isolation of Leptospira is challenging, since the bacteria of this genus are susceptible to adverse environmental conditions and may not remain viable for extend periods in urine samples. This study attempted to develop and evaluate a simple and practical method to isolate leptospires from bovine urine samples. A culture medium for sample transport, named Leptospira Transport Medium (LTM), was described and validated using reference serovars of Leptospira spp. in addition to autochthonous strains isolated in Brazil. We evaluated LTM in the field, by collecting 215 urine samples from slaughtered cattle and immediately seeding them in LTM and Fletcher's medium, used as control. The cultures were sent to a laboratory within 10 days for further processing. Moreover, 16S PCR was also performed on the urine samples directly to detect Leptospira DNA. Using LTM enabled 52 isolates (24.2%) to be obtained in pure culture, and contamination was only observed in 15/215 samples (7.0%). Regarding the samples in Fletcher's medium, 10 (4.6%) isolates were obtained. With 16S PCR performed in the urine samples, 31 samples (14.4%) were determined to be positive. LTM was developed and used in a simple and practical way and can significantly improve the isolation of leptospires from urine samples, as well as being highly useful in remote areas, not only in Brazil but also in other countries where few easily accessible laboratories are available. Furthermore, LTM can be prepared by laboratories and provided to veterinarians and technicians for urine collection in the field. • A culture medium named Leptospira transport medium (LTM) was developed. • LTM was used in the field to transport urine samples from cattle for the isolation of leptospires. • The use of LTM dispensed any laboratory facilities and immediate processing of the samples. • With LTM, the isolation of leptospires can be improved, especially in remote areas. [ABSTRACT FROM AUTHOR]