Genetic diversity of P1/pathogenic Leptospira species hosted by bats worldwide.

INTRODUCTION: Bats are a diverse group of mammals that have unique features allowing them to act as reservoir hosts for several zoonotic pathogens such as Leptospira. Leptospires have been classified into pathogenic, intermediate, and saprophytic groups and more recently into clades P1, P2, S1, and S2, being all the most important pathogenic species related to leptospirosis included within the P1/pathogenic clade. Leptospira has been detected from bats in several regions worldwide; however, the diversity of leptospires harboured by bats is still unknown. AIM: The aim of the present study was to determine the genetic diversity of Leptospira spp. harboured by bats worldwide. METHODS: A systematic review was conducted on four databases to retrieve studies in which Leptospira was detected from bats. All studies were screened to retrieve all available Leptospira spp. 16S rRNA sequences from the GenBank database and data regarding their origin. Sequences obtained were compared with each other and reference sequences of Leptospira species and analysed through phylogenetic analysis. RESULTS: A total of 418 Leptospira spp. 16S rRNA sequences isolated from 55 bat species from 14 countries were retrieved from 15 selected manuscripts. From these, 417 sequences clustered within the P1/pathogenic group, and only one sequence clustered within the P2/intermediate group. Six major clades of P1/pathogenic Leptospira spp. were identified, three of them composed exclusively of sequences obtained from bats. CONCLUSION: We identified that bats harbour a great genetic diversity of Leptospira spp. that form part of the P1/pathogenic clade, some of which are closely related to leptospirosis-associated species. This finding contributes to the knowledge of the diversity of leptospires hosted by bats worldwide and reinforces the role of bats as reservoirs of P1/pathogenic Leptospira spp.

Daños renales en fetos de ratas Wistar infectadas con Leptospira canicola

RESUMEN Introducción: La leptospirosis es una de las zoonosis que en Cuba se presenta de forma endémica, es causante de pérdidas humanas, animales y económicas, por lo que es un problema de salud. Las ratas constituyen un vector transmisor de gran importancia. Trabajos experimentales para estudiar las alteraciones renales fetales en modelo de rata Wistar no se han realizado en Cuba. Objetivo: Identificar las principales alteraciones morfométricas renales en fetos de ratas Wistar infectadas con serovariedad Leptospira canicola, en la Universidad de Ciencias Médicas de Pinar del Rio en el período comprendido entre 2017-2019. Método: Se realizó un estudio experimental en ratas Wistar preñadas: 2 grupos con tres animales cada uno: el grupo experimental con dosis de una concentración bacteriana de 300 millones de leptospiras por mililitros; se inoculó 1 ml a cada una infectándolas con cepas de Leptospira canicola. Se estudiaron los riñones de fetos extraídos por cesárea y se analizaron las variables: longitud renal y peso del riñón. Resultados: Los resultados obtenidos confirmaron los daños renales producidos por la Leptospira canicola tanto en el eje mayor como el menor de ambos riñones, así como variaciones en el peso de las crías infectadas. Conclusiones: La infección por Leptospira canicola en ratas Wistar preñadas provoca alteraciones en los riñones de los fetos lo que causa cambios en la morfometría de los ejes tanto mayor como menor, así como en el peso corporal del feto, lo que conlleva a una disminución del peso renal.

ABSTRACT Introduction: Leptospirosis is one of the endemic zoonoses in Cuba, causing human, animal and economic losses, making it a significant health problem. Rats are a very remarkable vector. Experimental work to study fetal renal alterations in the Wistar rats has not been carried out in Cuba. Objective: To identify the main renal morphometric alterations in fetuses of Wistar rats infected with serovar Leptospira canicola, at the Universidad de Ciencias Médicas de Pinar del Rio in the period between 2017-2019. Method: An experimental study was carried out in pregnant Wistar rats: 2 groups with three animals each: the experimental group with a dose of bacterial concentration of 300 million leptospires per milliliter; 1 ml was inoculated to each one, infecting them with strains of Leptospira canicola. The kidneys of fetuses extracted by cesarean section were studied, and the variables: renal length and kidney weight were analyzed. Results: The results obtained confirmed kidney damage caused by Leptospira canicola both in the major and minor axis of both kidneys, as well as variations in the weight of the infected pups. Conclusions: Leptospiral infection in pregnant Wistar rats causes alterations in the kidneys of the fetuses, bringing changes in the morphometry of both the major and minor axes, as well as in the body weight of the fetus, which leads to a decrease in renal weight.

RESUMO Introdução: A leptospirose é uma das zoonoses endêmicas em Cuba, causando prejuízos humanos, animais e econômicos, tornando-se um problema de saúde. Os ratos são um vetor transmissor muito importante. Trabalho experimental para estudar as alterações renais fetais no modelo de rato Wistar não foi realizado em Cuba. Objetivo: Identificar as principais alterações morfométricas renais em fetos de ratos Wistar infectados com sorovar Leptospira canicola, na Universidad de Ciencias Médicas de Pinar del Rio no período entre 2017-2019. Método: Estudo experimental realizado em ratas Wistar prenhes: 2 grupos com três animais cada: o grupo experimental com dose de concentração bacteriana de 300 milhões de leptospiras por mililitro; 1 ml fora inoculado em cada um, infectando-os com cepas de Leptospira canicola. Foram estudados os rins de fetos extraídos por cesariana e analisadas as variáveis: comprimento renal e peso renal. Resultados: Os resultados obtidos confirmaram o dano renal causado por Leptospira canicola tanto no eixo maior quanto no eixo menor de ambos os rins, bem como variações no peso dos filhotes infectados. Conclusões: A infecção por Leptospira canicola em ratas Wistar prenhes causa alterações nos rins dos fetos, o que provoca alterações na morfometria dos eixos maior e menor, bem como no peso corporal do feto, o que leva a uma diminuição da peso do rim.

Leptospirosis in dairy cattle from southern Brazil - risk factors

Background: Cattle are susceptible to chronic leptospirosis infection, that results in reduced milk production and reproductive disorders such as abortions, stillbirths, fetal malformation, and mummified fetuses, causing significant economic losses.Commercially available vaccines against leptospirosis offer limited protection to cattle because they contain only the mostprevalent serovars worldwide, even though they are not prevalent in the specific region. This study aimed to evaluate theprevalence of specific antibodies against Leptospira serogroups, reproductive disorders and the risk factors in dairy herdsfrom different mesoregions of Rio Grande do Sul State, Southern Brazil.Materials, Methods & Results: An epidemiological survey was conducted, and serum samples from the bovine population representative of three mesoregions (MR1, MR2, and MR3) were studied; the samples were collected and tested forleptospirosis using the microscopic agglutination test (MAT) for 12 serogroups checking for the presence of agglutination.A total of 442 blood samples were collected from dairy cattle from November to December 2019 (MR1, 187; MR2, 88;and MR3, 167), including cows vaccinated with different commercial vaccines during the three months before sample collection (n = 295) and non-vaccinated against leptospirosis (n = 147). At the time of collection, an interview was conductedwith the owners with questions about the health of the animals, management, habitat, feeding and reproduction. Chi-squaretests univariate analysis with the SPSS® version 20.0 were performed to estimate the association of serogroup Djasimanseroreactivity with the occurrence of reproductive problems and related risk factors. The mean prevalence of antibodiesagainst leptospires was 78.7% (MR1, 74.9 %; MR2, 84.1 %; and MR3, 80.2 %). Serogroup prevalence was different in...

Leptospira spp. of the urinary tract of female carrier goats in semi-arid conditions

Background: Leptospirosis is an important infectious disease in goat farming, with a worldwide distribution. It is usually transmitted by rodents and the genital route, may cause reproductive losses, negatively impacting goat farming. The diagnosis lies on serological, molecular and isolation techniques. Considering the importance of this disease for small ruminants, this work aimed to evaluate the serological, molecular findings and isolation of pathogenic leptospires in the urinary tract (kidney and bladder tissues) of goats. Materials, Methods & Results: Thirty-four adult goats were used for slaughter. Renal samples (n = 34), bladder (n = 34), were collected for isolation of the agent and molecular detection of Leptospira sp. and blood samples (n = 34) for serological testing. The polymerase chain reaction (PCR) was used as a molecular test and the microscopic serum agglutination test (MAT) was used as a serological test. Samples with DNA amplification were subjected to genetic sequencing. The presence of Leptospira DNA was found in the tissues of 8 (23.4%) goats, and of these, only 2 were positive in PCR and MAT. There was a slight agreement between the PCR and MAT techniques (k = 0.150; P = 0.436). In 6 (17.6%) samples of renal tissue and 2 (5.8%) bladder samples, Leptospira DNA was detected. The genes in a kidney tissue sample were sequenced and demonstrated 99% similarity to Leptospira interrogans. Anti-Leptospira sp. were detected in 6 (17.6%) of the animals tested. Discussion: Serology identified 3 predominant serogroups: Icterohaemorrhagiae, Tarassovi and Autumnalis, serogroups that are related to the presence of rodents that coexist in rural environments. Autumnalis has been reported in small ruminants, raising the hypothesis that goats are adapted, becoming chronic carriers and possible maintenance hosts. The frequency obtained (17.6%) may be the result of the mixed breed pattern and rustic characteristics inherent to the goat species. Given the characteristics of the semi-arid region, such as low rainfall and high solar incidence, it is essential to use an adapted methodology, with a lower cut-off point (1:50), as the serological titer is an established relationship between the animal species, the level of exposure throughout its evolution and the region studied. Molecular findings and bacterial isolation reveal the agent's ability to colonize the urinary tract of goats. These data show the importance that urine has in the epidemiological chain, being able to transmit the agent through direct contact with this product or through contamination of soil and water. There was no statistical agreement between the diagnostic techniques used in this study, in this case, an association between PCR and MAT is recommended to obtain data with high sensitivity and specificity. A bladder sample was sequenced and showed 99% similarity to Leptospira interrogans. In the semiarid region, the most common form of leptospirosis spread is through the sale of animals in business fairs for breeding, rearing or slaughter, as well as sharing the same property with several breeders. The introduction of chronic and asymptomatic carriers on the properties represents a serious risk for the spread of the disease. The results show the presence of Leptospira spp. in semi-arid goat herds, having as risk factors the presence of rodents and intercropping. The association of MAT and PCR is necessary for a better diagnosis of the disease.

Detección de Leptospira spp. (Spirochaetales: Leptospiraceae) en muestras ambientales de regiones habitadas por poblaciones vulnerables del norte argentino / Leptospira spp. (Spirochaetales: Leptospiraceae) detection in environmental samples from geographical regions inhabited by vulnerable communities in northern Argentina

Resumen La leptospirosis continúa siendo hoy en día un problema para la salud pública, principalmente en poblaciones de bajos recursos socioeconómicos. En este trabajo se presenta la detección de leptospiras patógenas en muestras ambientales (aguas y barros) provenientes de regiones del norte argentino (provincias de Formosa, Salta, Santiago del Estero, Misiones y Chaco) con variadas características climatológicas habitadas por poblaciones vulnerables. De las 89 muestras analizadas, en el 24,7% fue posible detectar molecularmente la presencia de leptospiras patógenas. La prevalencia por tipo de muestra fue de 27,8% para las aguas y 11,8% para los barros. Todas las localidades muestreadas presentaron al menos una muestra positiva a alguna de las pruebas realizadas, por lo que el presente trabajo refleja la necesidad de profundizar los estudios de la leptospirosis en distintas regiones de la Argentina.

Abstract Leptospirosis remains as a major public health problem nowadays, mainly affecting vulnerable communities with low socioeconomic resources. In this study, the molecular detection of pathogenic leptospires from environmental samples (water and mud) from northern Argentina (Formosa, Salta, Santiago del Estero, Misiones and Chaco provinces) is described. Samples were obtained from regions with varied climatological features, all inhabited by vulnerable communities. From the 89 samples that were analyzed, 24.7% showed molecular evidence of the presence of pathogenic leptospires. Prevalence by sample type was: 27.8% in water samples and 11.8% in mud samples. All the sampled regions showed at least one positive sample. This result highlights the need of further research regarding leptospirosis in different regions of Argentina.

Comparison of three protocols to preserve Leptospira spp. in cat urine for efficient DNA extraction and PCR amplification

Background: The pathogenic leptospira infection in mammalian species can cause a range of acute or chronic manifestations and may result in a carrier state. Previous studies have suggested that cats were resistant to acute leptospirosis however, the description of some clinical cases suggests that Leptospira spp. may also be pathogenic to this species. Recentstudies have shown that leptospires may be shed in the urine of infected cats. Endogenous substances present in urine mayinhibit PCR and allow leptospires to evade detection. This study aims to compare three protocols for sample processingto optimize the detection of pathogenic leptospires in cat urine.Materials, Methods & Results: Three protocols to optimize the detection of pathogenic leptospires in cat urine were tested.Aliquots of standard concentration of L. interrogans serovar Canicola culture were added to urine samples to achieveconcentrations of 1×105 to 1×102 leptospires/mL for each protocol. In protocols A and B the urine was neutralized by theaddition of phosphate-buffered saline (PBS), pH 7.4, in a proportion of 1 PBS: 2.5 urine (v/v). In protocol A, PBS wasadded to neutralize the urine pH for the leptospiral organisms immediately after addition of leptospires. In protocol B,PBS was added just before DNA extraction. In protocol C, no PBS was added. DNA extraction was performed at 4, 24and 48 h after addition of the leptospires using a modified protocol. Samples were incubated at 37ºC for 10 min. Sampleswere then centrifuged (850 g) for 15 min, at 25ºC. The supernatants were transferred to another tube, and the pellets werediscarded. The supernatants were centrifuged (16060 g) for 20 min at 4ºC. The supernatants were then discarded, and thepellets resuspended and washed with 1000 µL of PBS. All the samples were centrifuged at 16060 g for an additional 20min at 25ºC. The supernatants were discarded and the pellets were resuspended in 100 µL of PBS and incubated at 94ºCfor...(AU)

Comparison of three protocols to preserve Leptospira spp. in cat urine for efficient DNA extraction and PCR amplification

Background: The pathogenic leptospira infection in mammalian species can cause a range of acute or chronic manifestations and may result in a carrier state. Previous studies have suggested that cats were resistant to acute leptospirosis however, the description of some clinical cases suggests that Leptospira spp. may also be pathogenic to this species. Recentstudies have shown that leptospires may be shed in the urine of infected cats. Endogenous substances present in urine mayinhibit PCR and allow leptospires to evade detection. This study aims to compare three protocols for sample processingto optimize the detection of pathogenic leptospires in cat urine.Materials, Methods & Results: Three protocols to optimize the detection of pathogenic leptospires in cat urine were tested.Aliquots of standard concentration of L. interrogans serovar Canicola culture were added to urine samples to achieveconcentrations of 1×105 to 1×102 leptospires/mL for each protocol. In protocols A and B the urine was neutralized by theaddition of phosphate-buffered saline (PBS), pH 7.4, in a proportion of 1 PBS: 2.5 urine (v/v). In protocol A, PBS wasadded to neutralize the urine pH for the leptospiral organisms immediately after addition of leptospires. In protocol B,PBS was added just before DNA extraction. In protocol C, no PBS was added. DNA extraction was performed at 4, 24and 48 h after addition of the leptospires using a modified protocol. Samples were incubated at 37ºC for 10 min. Sampleswere then centrifuged (850 g) for 15 min, at 25ºC. The supernatants were transferred to another tube, and the pellets werediscarded. The supernatants were centrifuged (16060 g) for 20 min at 4ºC. The supernatants were then discarded, and thepellets resuspended and washed with 1000 µL of PBS. All the samples were centrifuged at 16060 g for an additional 20min at 25ºC. The supernatants were discarded and the pellets were resuspended in 100 µL of PBS and incubated at 94ºCfor...

Pesquisa de sorovares de leptospiras em rebanho bovino leiteiro no estado do Piauí, Brasil / Survey of leptospira serovars in a dairy cattle herd in Piauí state, Brazil

Este trabalho teve como objetivo relatar a pesquisa de sorovares de leptospiras em animais de uma fazenda localizada no Município de Buriti dos Lopes, integrante da Bacia Leiteira de Parnaíba, Piauí, Brasil. Foram colhidas amostras de sangue de 18 vacas da raça Girolando que abortaram no período de julho de 2004 a abril de 2005. As amostras foram submetidas ao teste de soroaglutinação microscópica (SAM) utilizando-se antígenos vivos de 23 sorovares de leptospiras patogênicas. 83,33% (15/18) das amostras apresentaram aglutininas anti-leptospira, sendo 53,33% de sorovar Hardjoprajitno (8/15), 6,67% para Hardjobovis (1/15), Wolffi (1/15) e Shermani (1/15) e quatro (4/15) co-agluitinações. A amostra do reprodutor apresentou aglutininas antileptospira para o sorovar Hardjobovis com titulação 1:400 o que sugere que este animal teve participação na disseminação da infecção no rebanho.

This study aimed to report the leptospira serovar varieties in animals from a farm located in the county of Buriti dos Lopes, within the so-called "milk basin" of Parnaíba, state of Piauí, Brazil. Samples of blood were collected from 18 Girolando cows who aborted between July 2004 and April 2005, and a sample was taken from a breeder bull. The samples were subjected to the microscopic agglutination test (SAM), using live antigens of 23 pathogenic leptospira serovars. Serum samples were screened in the dilution of 1:100. Data analysis revealed that 83.33% (15/18) of the samples showed anti-leptospira agglutinins, of which 53.33% were for the Hardjoprajitno serovar (8/15), 6.67% each for Hardjobovis (1/15), Wolffi (1/15) and Shermani (1/15), along with 4 (4/15) coagglutinations. The breeder-bull sample showed anti-leptospira agglutinins for serovar Hardjobovis with titration of 1:400, suggesting that this animal had participated in the spread of the infection in the herd.

PESQUISA DE SOROVARES DE LEPTOSPIRAS EM REBANHO BOVINO LEITEIRO NO ESTADO DO PIAUÍ, BRASIL

ABSTRACT This study aimed to report the leptospira serovar varieties in animals from a farm located in the county of Buriti dos Lopes, within the so-called milk basin of Parnaíba, state of Piauí, Brazil. Samples of blood were collected from 18 Girolando cows who aborted between July 2004 and April 2005, and a sample was taken from a breeder bull. The samples were subjected to the microscopic agglutination test (SAM), using live antigens of 23 pathogenic leptospira serovars. Serum samples were screened in the dilution of 1:100. Data analysis revealed that 83.33% (15/18) of the samples showed anti-leptospira agglutinins, of which 53.33% were for the Hardjoprajitno serovar (8/15), 6.67% each for Hardjobovis (1/15), Wolffi (1/15) and Shermani (1/15), along with 4 (4/15) coagglutinations. The breeder-bull sample showed anti-leptospira agglutinins for serovar Hardjobovis with titration of 1:400, suggesting that this animal had participated in the spread of the infection in the herd.

RESUMO Este trabalho teve como objetivo relatar a pesquisa de sorovares de leptospiras em animais de uma fazenda localizada no Município de Buriti dos Lopes, integrante da Bacia Leiteira de Parnaíba, Piauí, Brasil. Foram colhidas amostras de sangue de 18 vacas da raça Girolando que abortaram no período de julho de 2004 a abril de 2005. As amostras foram submetidas ao teste de soroaglutinação microscópica (SAM) utilizando-se antígenos vivos de 23 sorovares de leptospiras patogênicas. 83,33% (15/18) das amostras apresentaram aglutininas anti-leptospira, sendo 53,33% de sorovar Hardjoprajitno (8/15), 6,67% para Hardjobovis (1/15), Wolffi (1/15) e Shermani (1/15) e quatro (4/15) co-agluitinações. A amostra do reprodutor apresentou aglutininas antileptospira para o sorovar Hardjobovis com titulação 1:400 o que sugere que este animal teve participação na disseminação da infecção no rebanho.

EMJH medium with 5-fluorouracil and nalidixic acid associated with serial dilution technique used to recover Leptospira spp from experimentally contaminated bovine semen / Meio de EMJH com 5-fluorouracil e ácido nalidíxico associado a técnica das diluições seriadas usados para recuperar Leptospira spp do sêmen bovino experimentalmente contaminado

Bovine semen experimentally contaminated with Leptospira santarosai serovar Guaricura was submitted tothe modified EMJH medium with 5-fluorouracil (300mg/L) and nalidixic acid (20mg/L), named as ôselectivemediumõ and using the serial dilution technique, in order to evaluate the percentage of recovery of the addedmicroorganism. The selective EMJH medium was found with higher percentage of recovery of leptospirasand minor losses of samples due to contamination with opportunistic microorganisms than the non-selectiveEMJH medium: 151/376 (40.0%) of positive growth; and 38/376 (10.0%) contamination and 58/376 (15%) and129/376 (34.0%), respectively. These results were statistically significant (p<0. 0001; Fisher). Differences werefound when the frequencies of positive leptospires recovery have been compared in the serial dilution technique (10-1 to 10-4) between the selective and non-selective media at different dilution factors. At 1/10th dilution the percentages found were (0%, 0/80) and (38%, 30/80), at 1/100th dilution, (3%, 2/80) and (49%, 39/ 80) and at 1/1,000th dilution, (25%, 20/80) and (50%, 40/80), respectively. The percentage of recovery of leptospires was found to be directly proportional to the dilution used. The methodology of the serial dilution technique (setting at least three dilutions) and the use of selective EMJH medium have been found to be efficient for the isolation of leptospires from the bovine semen samples.

Sêmen bovino experimentalmente contaminado com a estirpe Leptospira santarosai Sorovar Guaricura foi utilizado para verificar a porcentagem de recuperação de leptospiras, utilizandoo meio de EMJH com 5-fluorouracil (300 mg/L) e ácido nalidíxico (20 mg/L), denominado ômeio seletivoõ, associado à técnica das diluições seriadas. O meio seletivo EMJH foi encontrado comporcentagem de recuperação mais elevada e com menos perda de amostras devido à contaminação com microorganismos oportunistas quando comparado com o meio EMJH não seletivo, que foram encontrados, respectivamente, com 151/376 (40.0%) de crescimento positivo e 38/376 (10.0%) de contaminação, 58/376 (15%) e 129/376 (34.0%). Estes resultados foram estatíticamente significantes (p<0, 0001; Fisher). Diferenças foram encontradas quando as freqüências de leptospiras recuperadas foram comparadas com a técnica de diluição seriada (10-1 a 10-4) e entre os meios seletivo e não-seletivo, em diferentes níveis de diluição. À diluição de 1/10, as porcentagens encontradas foram (0%, 0/80) e (38%, 30/80); à diluição 1/100, (3%, 2/80) e (49%, 39/80); e à diluição 1/1000, (25%, 20/80) e (50%, 40/80), respectivamente. A porcentagem de recuperação de leptospiras foi diretamente proporcional às diluições empregadas. A metodologia das diluições seriadas (estabelecendo pelo menos três diluições) e o uso de meio EMJH seletivo mostraram ser eficientes no isolamento de leptospiras presentes nas amostras de sêmen bovino.

EMJH medium with 5-fluorouracil and nalidixic acid associated with serial dilution technique used to recover Leptospira spp from experimentally contaminated bovine semen.

Bovine semen experimentally contaminated with Leptospira santarosai serovar Guaricura was submitted to the modified EMJH medium with 5-fluorouracil (300mg/L) and nalidixic acid (20mg/L), named as "selective medium" and using the serial dilution technique, in order to evaluate the percentage of recovery of the added microorganism. The selective EMJH medium was found with higher percentage of recovery of leptospiras and minor losses of samples due to contamination with opportunistic microorganisms than the non-selective EMJH medium: 151/376 (40.0%) of positive growth; and 38/376 (10.0%) contamination and 58/376 (15%) and 129/376 (34.0%), respectively. These results were statistically significant (p<0. 0001; Fisher). Differences were found when the frequencies of positive leptospires recovery have been compared in the serial dilution technique (10(-1) to 10(-4)) between the selective and non-selective media at different dilution factors. At 1/10(th) dilution the percentages found were (0%, 0/80) and (38%, 30/80), at 1/100(th) dilution, (3%, 2/80) and (49%, 39/ 80) and at 1/1,000(th) dilution, (25%, 20/80) and (50%, 40/80), respectively. The percentage of recovery of leptospires was found to be directly proportional to the dilution used. The methodology of the serial dilution technique (setting at least three dilutions) and the use of selective EMJH medium have been found to be efficient for the isolation of leptospires from the bovine semen samples.

EMJH medium with 5-fluorouracil and nalidixic acid associated with serial dilution technique used to recover Leptospira spp from experimentally contaminated bovine semen

Bovine semen experimentally contaminated with Leptospira santarosai serovar Guaricura was submitted to the modified EMJH medium with 5-fluorouracil (300mg/L) and nalidixic acid (20mg/L), named as "selective medium" and using the serial dilution technique, in order to evaluate the percentage of recovery of the added microorganism. The selective EMJH medium was found with higher percentage of recovery of leptospiras and minor losses of samples due to contamination with opportunistic microorganisms than the non-selective EMJH medium: 151/376 (40.0%) of positive growth; and 38/376 (10.0%) contamination and 58/376 (15%) and 129/376 (34.0%), respectively. These results were statistically significant (p 0. 0001; Fisher). Differences were found when the frequencies of positive leptospires recovery have been compared in the serial dilution technique (10-1 to 10-4) between the selective and non-selective media at different dilution factors. At 1/10th dilution the percentages found were (0%, 0/80) and (38%, 30/80), at 1/100th dilution, (3%, 2/80) and (49%, 39/80) and at 1/1,000th dilution, (25%, 20/80) and (50%, 40/80), respectively. The percentage of recovery of leptospires was found to be directly proportional to the dilution used. The methodology of the serial dilution technique (setting at least three dilutions) and the use of selective EMJH medium have been found to be efficient for the isolation of leptospires from the bovine semen samples.

Sêmen bovino experimentalmente contaminado com a estirpe Leptospira santarosai Sorovar Guaricura foi utilizado para verificar a porcentagem de recuperação de leptospiras, utilizando o meio de EMJH com 5-fluorouracil (300 mg/L) e ácido nalidíxico (20 mg/L), denominado "meio seletivo", associado à técnica das diluições seriadas. O meio seletivo EMJH foi encontrado com porcentagem de recuperação mais elevada e com menos perda de amostras devido à contaminação com microorganismos oportunistas quando comparado com o meio EMJH não seletivo, que foram encontrados, respectivamente, com 151/376 (40.0%) de crescimento positivo e 38/376 (10.0%) de contaminação, 58/376 (15%) e 129/376 (34.0%). Estes resultados foram estatíticamente significantes (p 0, 0001; Fisher). Diferenças foram encontradas quando as freqüências de leptospiras recuperadas foram comparadas com a técnica de diluição seriada (10-1 a 10-4) e entre os meios seletivo e não-seletivo, em diferentes níveis de diluição. À diluição de 1/10, as porcentagens encontradas foram (0%, 0/80) e (38%, 30/80); à diluição 1/100, (3%, 2/80) e (49%, 39/80); e à diluição 1/1000, (25%, 20/80) e (50%, 40/80), respectivamente. A porcentagem de recuperação de leptospiras foi diretamente proporcional às diluições empregadas. A metodologia das diluições seriadas (estabelecendo pelo menos três diluições) e o uso de meio EMJH seletivo mostraram ser eficientes no isolamento de leptospiras presentes nas amostras de sêmen bovino.

Comparison of four antibiotics for inactivating leptospires in bull semen diluted in egg yolk extender and experimentally inoculated with Leptospira santarosai serovar guaricura

Inactivation of leptospires in pools of semen from three Holstein Friesian bulls, collected in an artificial vagina, was investigated. Spermatic concentration was adjusted in egg yolk citrate extender, submitted to the following treatments: A (control; without antibiotics); B (penicillin, 1,000 UI/mL - streptomycin, 1,000 µg/mL); C (amoxicillin, 1,000 µg/mL); D (ceptiofur sodium, 1,000 µg/mL); E (amoxicillin 1,000 µg/mL - ceptiofur sodium 1,000 µg/mL). Leptospires (2.0 x 10(6) leptospires/mL) were added into the diluted semen. Recovery of leptospires was obtained in modified EMJH semi-solid medium with and without antibiotics. The antibiotics in the concentrations used did not affect means of percentage of progressive motility and individual progressive motility of spermatozoids. Penicillin-streptomycin presented the best results in leptospire inactivation (97.1%). Amoxicillin, ceptiofur sodium and their combination at the concentrations studied presented poor results: 59.29%; 32.5% and 60.36% of inactivation, being less effective in leptospire inactivation than penicillin-streptomycin.

A inativação de leptospiras em misturas de sêmen, obtidas através de vagina artificial, de três touros holandeses, foi estudada. A concentração espermática foi ajustada em diluidor gema-citrato utilizando os seguintes tratamentos: A (controle; sem antibióticos); B (penicilina, 1000 UI/mL - estreptomicina, 1000 µg/mL); C (amoxicilina, 1000 µg/mL); D (ceftiofur sódico, 1000 µg/mL); E (amoxicilina 1000 µg/mL - ceftiofur sódico 1000 µg/mL). Leptospiras (2,0x10(6) leptospiras/mL) foram adicionadas ao sêmen diluído. A recuperação das leptospiras foi obtida em meio EMJH modificado semi-sólido, com e sem antibióticos. As médias da porcentagem de motilidade progressiva e a de motilidade individual progressiva dos espermatozóides não foram afetadas pelos antibióticos nas concentrações usadas. Penicilina-estreptomicina apresentou os melhores resultados na inativação das leptospiras (97.1%). Amoxicilina, ceftiofur sódico e suas combinações, nas concentrações estudadas, apresentaram resultados insatisfatórios: 59.29%; 32.5% e 60.36% de inativação, sendo menos efetivos na inativação das leptospiras do que penicilina-estreptomicina.