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.

Molecular and serological characterization of pathogenic Leptospira spp. isolated from symptomatic dogs in a highly endemic area, Brazil.

BACKGROUND: Leptospirosis is an endemic zoonosis in Brazil, with a great impact on human and animal health. Although dogs are frequently infected by pathogenic Leptospira, the current epidemiological understanding of canine leptospirosis is mainly based on serological tests that predict the infecting serogroup/serovar. Thus, the present study aimed at identifying the causative agent for severe cases of canine leptospirosis in a highly endemic area through the isolation and characterization of the isolated strains. RESULTS: Urine, serum and blood samples were collected from 31 dogs with suspected acute leptospirosis treated at the Veterinary Hospital Service of Santo Amaro University between 2018 and 2019. Acute infection was confirmed in 17 dogs (54.8%) by the associated use of Polymerase Chain Reaction (PCR), Microscopic Agglutination (MAT) and bacteriological culture. Eleven dogs (35.5%) had titers ≥800, with the most frequent serogroups being Autumnalis and Icterohaemorrhagiae (n = 4 each) and Canicola (n = 2). Leptospires were recovered from four dogs, and Multilocus Sequence Analysis (MLSA) revealed infection caused by L. interrogans, which were further characterized as serogroups Canicola (n = 1) and Icterohaemorrhagiae (n = 3). CONCLUSION: The identity of the isolates and serological pattern of MAT suggest that dogs are highly exposed to the serogroup Icterohaemorrhagiae and Canicola, also indicating possible circulation of serogroups not yet isolated in Brazil, notably serogroup Autumnalis. Our findings also reinforce the usefulness of using multiple diagnostic approaches to confirm acute canine leptospirosis.

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.

Canine leptospirosis in asymptomatic populations from the Southwest Region of São Paulo State, Brazil / Leptospirose canina em uma população assintomática da região sudoeste do estado de São Paulo, Brasil

Leptospirosis is an infectious disease that can affect animals and humans. Distributed worldwide, the disease is more prevalent in tropical regions due to socioenvironmental characteristics. Dogs can serve as sentinels for environmental contamination due to their frequent contact with humans. This study investigated the frequency of occurrence of canine leptospirosis in asymptomatic populations from the Southwest Region of the State of São Paulo, Brazil. Thus, blood samples collected from 572 asymptomatic dogs from the cities of Apiaí, Cananeia, Itapeva, and Itu were tested with a microscopic agglutination test (MAT). A total of 40.5% of animals in Apiaí reacted to Leptospira spp., 42.6% in Itapeva, 7.1% in Cananeia, and 5.1% in Itu. The data from the present study demonstrate that at least one animal from the municipalities of Itapeva, Apiaí, and Cananeia had a titer equal to or higher than 800, indicating that Leptospira is circulating in these municipalities and that the teams working on castration campaigns need to be educated on the correct use of personal protective equipment, especially when mechanically emptying the bladder of these animals. This study also suggests that castration campaigns can strategically monitor zoonotic diseases and assist in establishing preventive strategies for human and animal health.(AU)

A leptospirose é uma enfermidade infectocontagiosa que pode acometer os animais e o homem. Nos países tropicais e em desenvolvimento ocorrem 70% dos casos humanos, com mortalidade variando entre 10 a 70%. Os cães podem se tornar portadores assintomáticos por um longo período, podendo transmitir a Leptospira para humanos. Devido ao intenso convívio com o ser humano, os cães podem servir como sentinelas da contaminação ambiental. Esse trabalho investigou a frequência de ocorrência da leptospirose canina em populações assintomáticas da região sudoeste do estado de São Paulo. Para isso foram examinadas pela técnica de soroaglutinação microscópica (MAT), amostras de sangue provenientes de 572 cães assintomáticos dos municípios de Apiaí, Cananeia, Itapeva e Itu por amostragem de conveniência, oriundos de campanhas de castração. Em Apiaí, foram encontrados 40,5% dos animais reagentes para Leptospira spp.; em Itapeva, 42,6%; em Cananeia, 7,7% e em Itu, 5,1%. Os dados encontrados demonstram que, pelo menos, um animal dos municípios de Itapeva, Apiaí e Cananeia apresentaram título igual ou maior que 800, indicando a circulação da bactéria nessas localidades e que a equipe envolvida nas campanhas de castração precisam ser alertadas sobre o correto uso de equipamento de proteção individual, principalmente no esvaziamento mecânico da bexiga antes do procedimento cirúrgico. O estudo também sugere que as campanhas de castração podem ser estratégicas no monitoramento de doenças zoonóticas e poderiam auxiliar no estabelecimento de ações preventivas para a saúde humana e animal.(AU)

Frequency of anti-Leptospira spp. antibodies in dogs and wild small mammals from rural properties and conservation units in southern Brazil.

Leptospirosis is a disease of worldwide distribution that affects man and several animal species. Domestic and wild animals can behave as reservoirs of the agent. The aim of this study was to evaluate the frequency of anti-Leptospira spp. antibodies in dogs and wild small mammals from rural properties and conservation units of three municipalities of Rio Grande do Sul State, Brazil. Sera were collected from 192 domestic dogs and 132 small mammals, namely rodents of the Cricetidae and Caviidae families and marsupials of the Didelphidae family. The study used Microscopic Agglutination Test (MAT) against 23 serovars of Leptospira spp. Overall, 9.90% (19/192) dogs, and 1.51% (2/132) small mammals were seropositive. Leptospira interrogans serovar Australis was the most frequent serovar in dogs and antibodies against L. interrogans serovar Pomona were detected in a dog and a small mammal belonging to the same municipality. We conclude that in the regions studied the frequency of seropositivity in dogs is low, and leptospires do not seem to be circulating in small mammals, yet, further research is necessary to assess the real role that these animals may have for leptospirosis in the studied areas. This is the first serological survey in small mammals, including wild rodents, in the area that encompasses conservation units of great importance to Rio Grande do Sul state.

Development of a pooled antigen for use in the macroscopic slide agglutination test (MSAT) to detect Sejroe serogroup exposure in cattle.

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.

Circulating Leptospira species identified in cattle of the Brazilian Amazon.

The present study aimed to detect the most prevalent serogroups and circulating Leptospira species in cows from Brazilian Amazon. Samples of blood serum, urine and kidney of 208 animals were collected at a municipal slaughterhouse in the Baixo Tocantins region of Pará State, Northern Brazil. The tests used were microscopic agglutination test (MAT), bacteriological isolation, polymerase chain reaction (PCR) and DNA sequencing. The frequency of MAT-reactive cows was 46.6% (97/208) with titers ranging from 100 to 3200, being Sejroe serogroup the most prevalent. There was no Leptospira isolation, but the DNA of bacterium was detected in 5.8% (12/208) of the kidney and in 14.9% (31/208) of the urine samples. DNA sequencing was performed directly from PCR products of 30 samples (3 kidneys and 27 urines), with identification of four different species: L. borgpetersenii with 56.7% (17/30), followed by L. kirschneri with 13.3% (4/30), L. interrogans with 6.7% (2/30), L. santarosai with 3.3% (1/30), and 20.0% (6/30) of samples were identified only at the genus level. These results reveal a diversity and peculiarity for bovine leptospirosis in the Amazon region, mainly due to the low frequency of L. santarosai and more surprising, the presence of L. kirschneri, differently of what is observed in other regions of Brazil.