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...

Lipid Peroxidation and Antioxidant Enzymes Activity of Wistar Rats Experimentally Infected with Leptospira interrogans

Background: Leptospirosis is a zoonotic disease with world-wide distribution, caused by various serovars of Leptospira interrogans and is presumed to be the most widespread zoonosis. Hematological and biochemical changes associated with renal and hepatic pathology are commonly observed in leptospirosis. All leptospires are aerobes and therefore might be expected to generate peroxides during respiration. Enzymatic reduction of H 2 0 2 by leptospires has been reported by researchers. The pathogenesis may be related to direct effects of leptospiral compounds or inflammatory response due to oxidative stress. The present investigation was designed to study the lipid peroxidation and the activity of antioxidant enzymes in rats experimentally infected with L. interrogans . Materials, Methods & Results: Fifty four male adult rats (Wistar) specific pathogen free, weighing in average 200 grams were used. Rats were divided in nine groups, six animals each group, eight infected groups and one as not infected. Inoculation was performed intraperitoneally (Day 1), using different serovars of L. interrogans distributed by groups: hardjo (group A), wolffi (group B), grippotyphosa (group C), canicola (group D), Icterohaemorrhagiae (group E), bratislava (group F), pomona (group G) and butembo (group H) . Group I was composed by not-infected rats, serving as the negative control group. On day 15 PI all animals were anesthetized with isoflurane for blood collection and subsequently decapitated. Liver, spleen, kidney and brain were collected from all animals. Blood was allocated in tube without anticoagulant for serum acquisition to measurement of thiobarbituric acid reactive substances (TBARS). Lipid peroxidation (TBARS levels), superoxide dismutase (SOD), catalase (CAT) and non-protein thiols (NPSH) were measured in the liver, spleen and kidney, and TBARS were also evaluated in serum and brain. [...]

Lipid Peroxidation and Antioxidant Enzymes Activity of Wistar Rats Experimentally Infected with Leptospira interrogans

Background: Leptospirosis is a zoonotic disease with world-wide distribution, caused by various serovars of Leptospira interrogans and is presumed to be the most widespread zoonosis. Hematological and biochemical changes associated with renal and hepatic pathology are commonly observed in leptospirosis. All leptospires are aerobes and therefore might be expected to generate peroxides during respiration. Enzymatic reduction of H 2 0 2 by leptospires has been reported by researchers. The pathogenesis may be related to direct effects of leptospiral compounds or inflammatory response due to oxidative stress. The present investigation was designed to study the lipid peroxidation and the activity of antioxidant enzymes in rats experimentally infected with L. interrogans . Materials, Methods & Results: Fifty four male adult rats (Wistar) specific pathogen free, weighing in average 200 grams were used. Rats were divided in nine groups, six animals each group, eight infected groups and one as not infected. Inoculation was performed intraperitoneally (Day 1), using different serovars of L. interrogans distributed by groups: hardjo (group A), wolffi (group B), grippotyphosa (group C), canicola (group D), Icterohaemorrhagiae (group E), bratislava (group F), pomona (group G) and butembo (group H) . Group I was composed by not-infected rats, serving as the negative control group. On day 15 PI all animals were anesthetized with isoflurane for blood collection and subsequently decapitated. Liver, spleen, kidney and brain were collected from all animals. Blood was allocated in tube without anticoagulant for serum acquisition to measurement of thiobarbituric acid reactive substances (TBARS). Lipid peroxidation (TBARS levels), superoxide dismutase (SOD), catalase (CAT) and non-protein thiols (NPSH) were measured in the liver, spleen and kidney, and TBARS were also evaluated in serum and brain. [...](AU)

Leptospirose bovina: aumento na incidência da Leptospira interogans sorovar butembo no rebanho do estado de Santa Catarina, Brasil / Bovine leptospirosis: increased incidence of Leptospira interogans serovar butembo in the herd of the State of Santa Catarina, Brazil

Os problemas reprodutivos são as principais manifestações clínicas da leptospirose crônica em fêmeas bovinas, sendo freqüentemente os únicos sinais observados no rebanho. Leptospira interogans sorovar hardjo é o mais predominante e importante porque compromete o desempenho reprodutivo dos rebanhos acometidos, porém levantamentos sorológicos no Brasil têm revelado resultados variados quanto à ocorrência dos sorovares nesta espécie animal. O sorovar butembo, normalmente quando encontrado, está presente em pequena parcela dos resultados positivos, porém, nosso levantamento apontou um aumento na prevalência deste sorovar nas amostras recebidas e processadas no período de Janeiro de 2005 a Dezembro de 2008. Desta maneira o objetivo deste trabalho foi relatar um aumento na detecção do variante sorológico butembo em soros bovinos provenientes do estado de Santa Catarina, nas amostras analisadas pelo Laboratório de leptospirose (Lablepto) da Universidade Federal de Santa Maria (UFSM).

Reproductive problems are the main clinical manifestations of leptospirosis in cows and it is often the only signs observed in the herd. Leptospira Interogans serovar hardjo is the most prevalent and important because it affects the reproductive performance of herds, but serological surveys in Brazil have shown mixed results regarding the occurrence of serovars in animal species. The serovar butembo, usually when found, is present in small proportion of positive results, however, our survey indicated an increase in the prevalence of this serovar in the samples received and processed from January 2005 to December 2008. Thus, the objective of this study was to report an increase in the detection of serological variant butembo in bovine serum from the state of Santa Catarina, in the samples analyzed by the Laboratory of leptospirosis (Lablepto) from Universidade Federal de Santa Maria (UFSM).