Detection of toxins A/B and isolation of Clostridium difficile and Clostridium perfringens from dogs in Minas Gerais, Brazil

The objective of this study was to detect C. difficileA/B toxins and to isolate strains of C. perfringensand C. difficile from diarrheic and non-diarrheic dogs in Brazil. Stool samples were collected from 57 dogs, 35 of which were apparently healthy, and 22 of which were diarrheic. C. difficileA/B toxins were detected by ELISA, and C. perfringensand C. difficilewere identified by multiplex PCR. C. difficileA/B toxins were detected in 21 samples (36.8%). Of these, 16 (76.2%) were from diarrheic dogs, and five (23.8%) were from non-diarrheic dogs. Twelve C. difficile strains (21.1%) were isolated, of which ten were A+B+and two were A-B-. All non-toxigenic strains were isolated from non-diarrheic animals. The binary toxin gene cdtBwas found in one strain, which was A+B+and was derived from a non-diarrheic dog. C. perfringensstrains were isolated from 40 samples (70.2%). Of these, 18 (45%) were from the diarrheic group, and 22 (55%) belonged to the non-diarrheic group. All isolates were classified as C. perfringenstype A and there was an association between the detection of the cpegene and the presence of diarrhea. Interestingly, ten strains (25%) were positive for the presence of the cpb2gene. The high rate of detection of the A/B toxins in non-diarrheic dogs suggests the occurrence of subclinical disease in dogs or carriage of its toxins without disease. More studies are needed to elucidate the epidemiology of C. difficileand C. perfringensin dogs and to better our understanding of C. difficileas a zoonotic agent. This is the first study to report the binary toxin gene in C. difficilestrains isolated from dogs in Brazil.(AU)

Reduction in oxidative stress levels in the colonic mucosa without fecal stream after the application of enemas containing aqueous Ilex paraguariensis extract / Redução dos níveis de estresse oxidativo na mucosa cólica sem trânsito intestinal após aplicação de enemas contendo extrato aquoso de Ilex paraguariensis

PURPOSE: To evaluate the antioxidant effects of enemas containing aqueous extract of Ilex paraguariensis, comparing segments with and without fecal stream and correlating the segments with the duration of intervention. METHODS: Twenty-six Wistar rats were subjected to a diversion of the fecal stream in the left colon by a proximal colostomy and distal mucosal fistula. The rats were distributed randomly into two experimental groups of 13 animals each based on the time of sacrifice after surgical procedure (two or four weeks). Each group was then divided into two experimental subgroups that received either second daily enemas containing 0.9% saline solution or aqueous extract of Ilex paraguariensis at 0.2g/100g. Colitis was diagnosed by histopathological analysis and the detection of oxidative tissue damage by measuring the levels of malondialdehyde. The Mann-Whitney test was used to compare the tissue levels of malondialdehyde between colon segments with and without fecal stream in each experimental group, and the Kruskal-Wallis test was used to verify the variance between the levels of oxidative stress according the duration of the irrigation; both tests determined significance at 5% (p<0.05). RESULTS: The levels of malondialdehyde in the animals subjected to intervention in the colon with saline with and without fecal stream after two and four weeks of irrigation were 0.05±0.006 and 0.06±0.006, and 0.05± 0.03 and 0.08 ±0.02, respectively. The malondialdehyde levels in the animals irrigated with Ilex paraguariensis with and without fecal stream after two and four weeks of irrigation were 0.010±0.002 and 0.02±0.004, and 0.03±0.007 and 0.04±0.01, respectively. After two and four weeks of intervention, the levels of malondialdehyde were lower in the animals irrigated with Ilex paraguariensis regardless of the time of irrigation (p=0.0001 and p=0.002, respectively).(AU)

OBJETIVO: Avaliar os efeitos antioxidantes de clisteres contendo extrato aquoso de Ilex paraguariensis comparando segmentos com e sem trânsito fecal e correlacionando com o tempo de intervenção. MÉTODOS: Vinte e seis ratos Wistar machos foram submetidos à derivação do trânsito no cólon esquerdo pela confecção de colostomia proximal e fístula mucosa distal. Foram divididos de forma randomizada em dois grupos experimentais de 13 animais, de acordo com o sacrifício ser realizado após duas ou quatro semanas do procedimento cirúrgico. Cada grupo foi dividido em dois subgrupos experimentais segundo a aplicação diária ter sido realizada com enemas diários contendo solução fisiológica a 0,9% ou extrato aquoso de Ilex paraguariensis na concentração de 0,2 g/100 ml. O diagnóstico de colite foi estabelecido por análise histológica e os níveis de estresse oxidativo pela dosagem tecidual de malondialdeído Para comparar os níveis de malondialdeído entre os cólons com e sem trânsito fecal em cada grupo experimental adotou-se o teste de Mann-Withney, e o teste de Kruskal-Wallis para verificar a variação dos níveis de estresse oxidativo segundo o tempo de irrigação, estabelecendo-se para ambos o nível de significância de 5% (p<0,05). RESULTADOS: Os níveis de malondialdeído nos animais submetidos à intervenção com soro fisiológico nos cólons providos e desprovidos de trânsito fecal após duas e quatro semanas de irrigação foram de 0,05±0,006, 0,06±0,006 e 0,05±0,03, 0,08±0,02 respectivamente. Os níveis de malondialdeído nos animais irrigados com Ilex paraguariensis, nos cólons providos e desprovidos de trânsito, após duas e quatro semanas de irrigação foram de 0,010±0,002, 0,02±0,004 e 0,03±0,007, 0,04±0,01, respectivamente. Após duas e quatro semanas de intervenção os níveis de malondialdeído foram menores nos animais irrigados com Ilex paraguariensis, independente do tempo de irrigação (p=0,0001 e p=0,002, respectivamente).(AU)

The importance of oxygen free radicals in the etiopathogenesis of diversion colitis in rats / A importância dos radicais livres de oxigênio na etiopatogenia da colite de exclusão em ratos

PURPOSE: Quantify the levels of oxidative DNA damage of epithelial colon cells comparing segments with and without fecal stream. METHODS: Sixty Wistar rats were subjected to deviation of fecal stream by proximal colostomy and a distal mucosal fistula. Animals were divided into three experimental groups that were sacrificed 6, 12 and 24 weeks after surgery. In each experimental group, five animals underwent laparotomy without intestinal deviation (sham subgroup). The diagnosis of colitis was made by histopathological analysis and the inflammatory activity index by graduated scale. The neutrophil infiltration was determined by myeloperoxidase tissue levels and the intensity of oxidative DNA damage by comet assay. The Mann-Withney and Student t test were used to compare the results among experimental subgroups and the Kruskal-Wallis test for variance analysis, adopting a significance level of 5 percent (p<0.05). RESULTS: Colon segments without fecal stream was shown higher histological inflammatory score of the colon wall after 12 and 24 weeks (p=0.001) that increased with the time of diversion (p=0.01). The activity of myeloperoxidase in segments without fecal stream decreased with the time (p=0.001). Oxidative DNA damage levels were significantly higher in the segments without fecal stream, (p=0.0001), independent of time of colon diversion, and increase with the time (p=0.0007). CONCLUSIONS: Colon segments without fecal stream showed high levels of oxidative DNA damage related to histological alterations observed in diversion colitis. The levels of oxidative DNA damage in segments devoid of the fecal stream increase with the time of intestinal exclusion.(AU)

OBJETIVO: Quantificar os níveis de dano oxidativo ao DNA em células epiteliais da mucosa cólica comparando segmentos com e sem trânsito fecal. MÉTODOS: Sessenta ratos Wistar foram submetidos à derivação do trânsito intestinal por colostomia proximal e fístula mucosa distal. Os animais foram divididos em três grupos experimentais segundo terem sido sacrificados 6, 12 e 24 semanas após a cirurgia. Em cada grupo experimental, cinco animais foram submetidos à laparotomia isolada sem derivação fecal (grupo sham). O diagnóstico de colite foi estabelecido por análise histopatológica e o índice de atividade inflamatória por escala graduada. A infiltração neutrofílica foi determinada pelos níveis teciduais da mieloperoxidase e a intensidade do dano oxidativo ao DNA pelo ensaio em cometa. Utilizaram-se os testes de Mann-Withney e o teste t de Student para comparar os resultados encontrados entre os subgrupos experimentais e o teste de Kruskal-Wallis para análise de variância, adotando-se nível de significância de 5 por cento (p<0,05). RESULTADOS: Os segmentos cólicos, sem trânsito fecal apresentaram maior escore histológico de inflamação após 12 e 24 semanas (p=0,001), que aumentou com o tempo de derivação (p=0,01). A atividade da mieloperoxidase nos segmentos sem trânsito fecal diminuiu com o progredir do tempo (p=0,001). Os níveis de dano oxidativo ao DNA foram significativamente maiores nos segmentos sem trânsito fecal (p=0,0001), independente do tempo de exclusão considerado, aumentando com o progredir do tempo de exclusão (p = 0,0007). CONCLUSÕES: Segmentos cólicos desprovidos de trânsito fecal apresentam níveis elevados de dano oxidativo ao DNA relacionados às alterações histológicas observadas na colite de exclusão. Os níveis de dano oxidativo ao DNA nos segmentos desprovidos de trânsito fecal aumentam com o decorrer do tempo de exclusão.(AU)