Improvement of the enrichment used in the EMJH medium (Ellinghausen-McCullough-Johnson-Harris) for the cultivation of Leptospira spp.

Changes were made to the original formulation of the EMJH medium (Ellinghausen-McCullough-Johnson-Harris) enrichment and some aspects such as growth time of Leptospira and utilization in the microscopic agglutination test (MAT) were evaluated and compared to the original enrichment and to a commercially available enrichment (DIFCO™). Leptospira samples (24 antigens) that make up our panel of antigens used in MAT were used, among them, reference and autochthonous strains isolated in Brazil. The samples were grown individually in the EMJH medium under the three previously mentioned conditions (adapted enrichment, original enrichment and commercial enrichment). In addition, 89 blood serums from domestic and wild animals were analyzed by MAT using the antigens grown in these media. All samples tested grew efficiently with the adapted enrichment, and the MAT results were satisfactory. Therefore, other laboratories could also benefit from the use of this adapted enrichment when culturing the Leptospira strains applied in their MAT panels.

Isolation of an atypical Leptospira strain assigned to the Sejroe serogroup from a water buffalo in Brazil.

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.

Usefulness of the Ranking Technique in the Microscopic Agglutination Test (MAT) to Predict the Most Likely Infecting Serogroup of Leptospira.

The microscopic agglutination test (MAT) used for the serological diagnosis of leptospirosis, as a robust and inexpensive method, is still the reality in many laboratories worldwide. Both the performance and the interpretation of the MAT vary from region to region, making standardization difficult. The prediction of the probable infecting serogroup using this test is indispensable for elucidating the epidemiology of the disease; however, in veterinary medicine, many studies consider any reaction detected with a titer of 100, which may ultimately overestimate some serogroups. Thus, the aim of this study was to evaluate the usefulness of the ranking technique for predicting the probable infecting serogroup identified by the MAT, eliminating cross reactions with other serogroups. Leptospira strains (12 samples) were inoculated in hamsters, and after 30 days, serology was performed by the MAT for these animals to confirm the infecting serogroup. Using the ranking technique, the probable infectious serogroup found with the MAT was the same as that in which the strains of inoculated leptospires belonged; additionally, the technique can be applied in epidemiological studies involving herds.

Leptospira interrogans serogroup Pomona strains isolated from river buffaloes.

At present, little is known regarding the prevalence of buffalo leptospirosis worldwide, especially with respect to which Leptospira strains may infect this animal species. Furthermore, most investigations into this disease in buffaloes have only been performed with serological studies. In Brazil, particularly in the Amazon, buffalo production is growing and is just as important as cattle production, although few studies have been performed on buffalo compared to cattle. Thus, the aim of this study was to isolate and characterise Leptospira strains from river buffaloes raised in the Brazilian Amazon region. We collected 109 kidney samples from slaughtered buffaloes raised in the Amazon Delta region of Brazil. The samples were analysed by bacteriological culture for the isolation of leptospires, and the obtained isolates were serologically and molecularly characterised by microscopic agglutination test (MAT), DNA sequencing and multiple locus variable-number tandem repeat analysis (MLVA). Five isolates were obtained, and in serogrouping analyses, these isolates were only reactive for the Pomona serogroup, with an observed titre of 25,600. The DNA sequencing results revealed that all the isolates belonged to the species Leptospira interrogans, and the MLVA results showed that the VNTR loci 4, 7 and 10 profile of all the isolates was 4-1-10. In this study, we observed that Pomona serogroup strains circulate in buffaloes in the Amazon, showing that in Brazil, buffaloes can be affected by Leptospira strains other than the Sejroe group, which are adapted to cattle.

Leptospira strains isolated from cattle in the Amazon region, Brazil, evidence of a variety of species and serogroups with a high frequency of the Sejroe serogroup.

In Brazil, there have been few leptospires isolated from cattle, especially in the Amazon, implying that the epidemiology of the disease in this region is still largely unclear. In a previous study, 52 Leptospira isolates were obtained from urine of cattle raised in the Brazilian Amazon and, to achieve a greater understanding of Leptospira infection in cattle of this region, the present study aimed to serologically and molecularly characterizes all these isolates. The laboratory assays used were the microscopic agglutination test (MAT) adopting a panel of polyclonal antisera against Leptospira spp. for serogrouping the isolates, DNA sequencing (secY) and multiple locus variable number tandem repeat analysis (MLVA). The isolates belonged to five species: 20/52 were identified as L. borgpetersenii (38.5 %); 18/52 as L. kirschneri (34.6 %); 9/52 as L. santarosai (17.3 %); 3/52 as L. noguchii (5.8 %) and 2/52 as L. interrogans (3.8 %). With serogrouping, nine different serogroups were detected, with a high frequency of the Sejroe serogroup. MLVA showed that all L. borgpetersenii isolates had a profile compatible with serovar Hardjo; moreover, the other isolates demonstrated a diversity of patterns, and some of them may represent strains not yet characterized. In the Brazilian Amazon, the leptospires circulating in cattle revealed the unique aspects of infections in this area which, in addition to a variety of strains, were characterized by a high frequency of the Sejroe serogroup, highlighting the serovar Hardjo, which has not been reported in other regions of Brazil.

Leptospira transport medium (LTM): A practical tool for leptospires isolation.

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.

Identification of Pathogenic Leptospira Species in the Urogenital Tract of Water Buffaloes (Bubalus bubalis) From the Amazon River Delta Region, Brazil.

In the current context of deforestation and fire in the Amazon, buffaloes could be a cost-effective and sustainable alternative for cattle production in the region, as they can convert low-quality foods and be raised in floodplain areas. However, little is known about the reproductive diseases that affect these animals; thus, the purpose of this study was to perform the molecular characterization of Leptospira spp. in the urogenital tract of water buffaloes (Bubalus bubalis) raised in the Amazon River Delta region in Brazil. Samples were collected from 114 kidneys, 204 ovaries, and 160 uterine swabs of slaughtered buffaloes in the Macapá microregion of Amapá State (Brazil) and were subjected to PCR to detect bacterial DNA. Positive amplicons were sequenced to identify Leptospira species. Among the total samples, 11/473 were PCR positive (2.3%), including 10 kidney samples and one uterine swab sample. DNA sequencing identified two pathogenic species from the kidney samples: L. interrogans, accounting for 60.0% (6/10) of these samples, and L. borgpetersenii, accounting for 20.0% (2/10), while 20.0% (2/10) were identified only at the genus level. The bacterium in the uterine swab sample was identified as L. interrogans with genetic proximity to strains belonging to the serovar Hardjo. This is the first report of leptospires species identified in buffaloes from the Amazon River Delta region and revealed that these animals may be carriers of different pathogenic Leptospira species, similar to bovines, including showing genital colonization.