Histological analysis of the intestinal wall of newborn rats submitted to hypoxia and reoxygenation to evaluate the protective effect of N-Acetylcysteine

Purpose To evaluate the effect of N-Acetylcysteine (NAC) in newborn rats submitted to hypoxia and reoxygenation (H/R) conditions in an experimental model of necrotizing enterocolitis. Methods Eight pregnant rats and their 70 cubs were used (5 groups) and exposed to H/R conditions and received NAC at different times. The animals in the H/R groups were placed in a gas chamber (100% CO2) for 10 minutes and then reoxygenated for 10 minutes (100% O2), twice a day for the first three days of life, with a six-hour span between events. On the third day of life, the animals were anesthetized, laparotomized and the intestines were resected. Results The H/R and NAC groups showed changes in the intestinal wall in relation to the number, height and width of the villi when compared to the control group (p 0.0001), but with better preservation of structures in the NAC group. There were no differences between groups regarding the number (%) of mitoses. Conclusion The administration of NAC decreased the lesions in the intestinal wall of rats submitted to H/R, therefore suggesting that this drug can be used to prevent the development of necrotizing enterocolitis in newborns.(AU)

Clostridium perfringens α and ß recombinant toxoids in equine immunization / Toxóides recombinantes α e ß de Clostridium perfringens na imunização de equinos

Clostridium perfringens is considered one of the main causative agents of superacute enterocolitis, usually fatal in the equine species, due to the action of the ß toxin, and is responsible for causing severe myonecrosis, by the action of the α toxin. The great importance of this agent in the equine economy is due to high mortality and lack of vaccines, which are the main form of prevention, which guarantee the immunization of this animal species. The aim of this study was to evaluate three different concentrations (100, 200 and 400µg) of C. perfringens α and ß recombinant toxoids in equine immunization and to compare with a group vaccinated with a commercial toxoid. The commercial vaccine was not able to stimulate an immune response and the recombinant vaccine was able to induce satisfactory humoral immune response in vaccinated horses, proving to be an alternative prophylactic for C. perfringens infection.(AU)

Clostridium perfringens é considerado um dos principais agentes causadores de enterocolites superagudas, geralmente fatais na espécie equina, devido à ação da toxina ß, além de ser responsável por causar quadros graves de mionecrose, pela ação da toxina α. A grande importância desses agentes na equinocultura, deve-se a elevada mortalidade e a inexistência de vacinas, principal forma de prevenção, que garantam a imunização dessa espécie animal. O objetivo deste trabalho foi avaliar três diferentes concentrações (100, 200 e 400µg) dos toxóides recombinantes α e ß de C. perfringens na imunização de equinos, bem como comparar com um grupo vacinado com um toxóide comercial. A vacina comercial não se mostrou capaz de estimular uma resposta imune e a vacina recombinante foi capaz de induzir resposta imune humoral satisfatória em equinos vacinados, provando ser uma alternativa profilática para infecção por C. Perfringens.(AU)

Clostridium perfringens α and ß recombinant toxoids in equine immunization / Toxóides recombinantes α e ß de Clostridium perfringens na imunização de equinos

Clostridium perfringens is considered one of the main causative agents of superacute enterocolitis, usually fatal in the equine species, due to the action of the ß toxin, and is responsible for causing severe myonecrosis, by the action of the α toxin. The great importance of this agent in the equine economy is due to high mortality and lack of vaccines, which are the main form of prevention, which guarantee the immunization of this animal species. The aim of this study was to evaluate three different concentrations (100, 200 and 400µg) of C. perfringens α and ß recombinant toxoids in equine immunization and to compare with a group vaccinated with a commercial toxoid. The commercial vaccine was not able to stimulate an immune response and the recombinant vaccine was able to induce satisfactory humoral immune response in vaccinated horses, proving to be an alternative prophylactic for C. perfringens infection.(AU)

Clostridium perfringens é considerado um dos principais agentes causadores de enterocolites superagudas, geralmente fatais na espécie equina, devido à ação da toxina ß, além de ser responsável por causar quadros graves de mionecrose, pela ação da toxina α. A grande importância desses agentes na equinocultura, deve-se a elevada mortalidade e a inexistência de vacinas, principal forma de prevenção, que garantam a imunização dessa espécie animal. O objetivo deste trabalho foi avaliar três diferentes concentrações (100, 200 e 400µg) dos toxóides recombinantes α e ß de C. perfringens na imunização de equinos, bem como comparar com um grupo vacinado com um toxóide comercial. A vacina comercial não se mostrou capaz de estimular uma resposta imune e a vacina recombinante foi capaz de induzir resposta imune humoral satisfatória em equinos vacinados, provando ser uma alternativa profilática para infecção por C. Perfringens.(AU)

Infectious diseases dynamics in growing/finishing pigs in Southern Brazil (2005-2016) / Dinâmica de ocorrência das doenças infecciosas em suínos nas fases de crescimento e terminação no sul do Brasil (2005-2016)

This study aimed to determine the frequency and distribution of infectious diseases diagnosed through necropsy examination and histopathological analysis in growing/finishing pigs along 12 years (2005-2016) in Southern Brazil. We evaluated 1906 anatomopathological exams of pigs at growing/finishing phases, of which the infectious diseases corresponded to 75.6% of the cases (1,441/1,906). Porcine circovirus type 2 (PCV2) infections were the most frequent, accounting for 51.3% of the cases (739/1,441) with a higher frequency from 2005 to 2007, characterizing an epidemic distribution, with a gradual decline after 2008. Infectious diseases affecting the respiratory system were the second major cause with 30.1% of the cases. Among these, necrotizing bronchiolitis caused by swine Influenza (15.1%, 218/1,441) and bacterial pneumonia (15%, 216/1,441) were the main conditions. Influenza was mostly diagnosed from 2010 to 2013, accounting for 43.1% (167/387) of the cases. After this period, both respiratory infectious diseases were endemic. Digestive system infectious diseases accounted for 10.5% of the diagnoses (151/1,441), with the following main conditions: Salmonella spp. enterocolitis (43.7%, 66/151), Lawsonia spp. proliferative enteropathy (41.7%, 63/151), and Brachyspira spp. colitis (14.6%, 22/151). The latter had a higher incidence from 2012 to 2014 with all cases detected in this period. Polyserositis and bacterial meningitis represented, respectively, 5.8% (84/1,441) and 2.3% (33/1,441) of the cases diagnosed, with a constant endemic character.(AU)

O objetivo deste estudo consistiu em determinar a frequência e a distribuição das doenças infecciosas diagnosticadas através de exame de necropsia e análise histopatológica em suínos nas fases de crescimento/terminação ao longo de 12 anos (2005-2016) no sul do Brasil. Foram avaliados 1906 laudos anatomopatológicos de suínos nas fases de crescimento/terminação, dos quais as doenças infecciosas corresponderam a 75,6% (1441/1906) do total. As infecções por circovírus suíno tipo 2 (PCV2) foram as mais frequentes, contabilizando 51,3% (739/1441) dos casos, com uma alta frequência de 2005 a 2007 caracterizando uma distribuição epidêmica neste período, e um declínio gradual após o ano de 2008. A segunda principal causa incluiu as doenças infecciosas que afetam o sistema respiratório (30,1% dos casos). Dentre essas, destacaram-se a influenza suína (15,1%; 218/1441) e pneumonias bacterianas (15%; 216/1441). O diagnóstico de influenza apresentou uma frequência elevada de 2010 a 2013, totalizando 43,1% (167/387) dos casos. Após este período, ambas doenças infecciosas respiratórias exibiram caráter endêmico. As doenças infecciosas do sistema digestório totalizaram 10,5% (151/1441) dos diagnósticos, com as seguintes principais condições: enterocolite por Salmonella spp. (43,7%; 66/151), enteropatia proliferativa por Lawsonia spp. (41,7%; 63/151) e colite por Brachyspira spp. (14,6%; 22/151). A colite por Brachyspira spp. apresentou uma alta incidência de 2012 a 2014 com todos os casos detectados no período. As polisserosites e meningites bacterianas representaram 5,8% (84/1441) e 2,3% (33/1441) dos casos diagnosticados, respectivamente, com um caráter endêmico constante.(AU)

Infectious diseases dynamics in growing/finishing pigs in Southern Brazil (2005-2016) / Dinâmica de ocorrência das doenças infecciosas em suínos nas fases de crescimento e terminação no sul do Brasil (2005-2016)

This study aimed to determine the frequency and distribution of infectious diseases diagnosed through necropsy examination and histopathological analysis in growing/finishing pigs along 12 years (2005-2016) in Southern Brazil. We evaluated 1906 anatomopathological exams of pigs at growing/finishing phases, of which the infectious diseases corresponded to 75.6% of the cases (1,441/1,906). Porcine circovirus type 2 (PCV2) infections were the most frequent, accounting for 51.3% of the cases (739/1,441) with a higher frequency from 2005 to 2007, characterizing an epidemic distribution, with a gradual decline after 2008. Infectious diseases affecting the respiratory system were the second major cause with 30.1% of the cases. Among these, necrotizing bronchiolitis caused by swine Influenza (15.1%, 218/1,441) and bacterial pneumonia (15%, 216/1,441) were the main conditions. Influenza was mostly diagnosed from 2010 to 2013, accounting for 43.1% (167/387) of the cases. After this period, both respiratory infectious diseases were endemic. Digestive system infectious diseases accounted for 10.5% of the diagnoses (151/1,441), with the following main conditions: Salmonella spp. enterocolitis (43.7%, 66/151), Lawsonia spp. proliferative enteropathy (41.7%, 63/151), and Brachyspira spp. colitis (14.6%, 22/151). The latter had a higher incidence from 2012 to 2014 with all cases detected in this period. Polyserositis and bacterial meningitis represented, respectively, 5.8% (84/1,441) and 2.3% (33/1,441) of the cases diagnosed, with a constant endemic character.(AU)

O objetivo deste estudo consistiu em determinar a frequência e a distribuição das doenças infecciosas diagnosticadas através de exame de necropsia e análise histopatológica em suínos nas fases de crescimento/terminação ao longo de 12 anos (2005-2016) no sul do Brasil. Foram avaliados 1906 laudos anatomopatológicos de suínos nas fases de crescimento/terminação, dos quais as doenças infecciosas corresponderam a 75,6% (1441/1906) do total. As infecções por circovírus suíno tipo 2 (PCV2) foram as mais frequentes, contabilizando 51,3% (739/1441) dos casos, com uma alta frequência de 2005 a 2007 caracterizando uma distribuição epidêmica neste período, e um declínio gradual após o ano de 2008. A segunda principal causa incluiu as doenças infecciosas que afetam o sistema respiratório (30,1% dos casos). Dentre essas, destacaram-se a influenza suína (15,1%; 218/1441) e pneumonias bacterianas (15%; 216/1441). O diagnóstico de influenza apresentou uma frequência elevada de 2010 a 2013, totalizando 43,1% (167/387) dos casos. Após este período, ambas doenças infecciosas respiratórias exibiram caráter endêmico. As doenças infecciosas do sistema digestório totalizaram 10,5% (151/1441) dos diagnósticos, com as seguintes principais condições: enterocolite por Salmonella spp. (43,7%; 66/151), enteropatia proliferativa por Lawsonia spp. (41,7%; 63/151) e colite por Brachyspira spp. (14,6%; 22/151). A colite por Brachyspira spp. apresentou uma alta incidência de 2012 a 2014 com todos os casos detectados no período. As polisserosites e meningites bacterianas representaram 5,8% (84/1441) e 2,3% (33/1441) dos casos diagnosticados, respectivamente, com um caráter endêmico constante.(AU)

Oxidative stress assessment in intestine of newborn rats submitted to hypoxia and reoxygenation with tadalafil

Purpose: To evaluate the functional and structural response of tadalafil effects in the intestinal mucosa, using an experimental model of hypoxia and reoxygenation injury in rats. Methods: The animals were divided into 4 groups: CTL, H/R, H/R+Td and M+Td. The newborn rats allocated in groups H/R, H/R+Td and M+Td were submitted twice a day, to a gas chamber with CO2 at 100% for 10 minutes and afterward reoxygenation with O2 at 98% for 10 minutes, in the three first days of life. Tadalafil dose was given to newborn of group H/R+Td and to the pregnant rat of group M+Td. Histological analysis was made with hematoxylin-eosin technique and oxidative stress through nitrite and nitrate levels and lipid peroxidation. Results: The histological analysis showed a reduction of mucosa alterations in the groups that received tadalafil. In the oxidative stress evaluation, occurred an increase of NO levels and less lipidic peroxidation in the ileum segments that received tadalafil. Conclusion: Tadalafil provides tissue protection when administered independently to both, pregnant or newborns.(AU)

Putative equine neorickettsiosis in a mare from Southern Brazil

Background: This report describes the occurrence of equine neorickettsiosis (EN) in the northern region of Paraná,southern Brazil. EN is a non-contagious infectious disease caused by the Gram-negative bacterium, Neorickettsia risticii.Equine neorickettsiosis was previously known as Potomac horse fever and monocytic ehrlichiosis. The disease occurspredominantly in the USA and Canada; data relative to EN in Brazil is scarce. The aim of this study was to report the firstcase of putative EN in the state of Paraná due to a combination of IHC and molecular testing.Case: A 2-year-old Quarter Horse was referred to a Veterinary Hospital with episodes of abdominal discomfort, fever,anorexia, tachycardia, and tachypnea. The animal reportedly demonstrated episodes of blackened and fetid diarrhea afterthe ingestion of hay. A treatment was established upon arrival at the veterinary hospital, but the mare died after 12 hoursof monitoring. An autopsy examination performed soon after death revealed severe hyperemia of the mucosa of the cecum and colon, with multifocal cecal erosions and ulcerations. The principal histological lesion observed was necrotizingenterocolitis. Additional significant histopathologic lesions included widespread lymphoid depletion affecting the spleen,tonsils, and lymph nodes. An IHC assay designed to identify the antigens of N. helminthoeca (NH) in formalin fixed paraffin embedded (FFPE) tissues, identified antigens of intralesional neorickettsial organisms within macrophages of themucosa of the colon. Additionally, a PCR assay designed to amplify the 16S rRNA gene of Neorickettsia, amplified thedesired amplicon, but sequencing was frustrating.Discussion: A putative diagnosis of equine neorickettsiosis was established due to the combination of epidemiologicalevidence, pathologic findings, immunohistochemical identification of intralesional antigens of neorickettsial agents, andamplification...

Putative equine neorickettsiosis in a mare from Southern Brazil

Background: This report describes the occurrence of equine neorickettsiosis (EN) in the northern region of Paraná,southern Brazil. EN is a non-contagious infectious disease caused by the Gram-negative bacterium, Neorickettsia risticii.Equine neorickettsiosis was previously known as Potomac horse fever and monocytic ehrlichiosis. The disease occurspredominantly in the USA and Canada; data relative to EN in Brazil is scarce. The aim of this study was to report the firstcase of putative EN in the state of Paraná due to a combination of IHC and molecular testing.Case: A 2-year-old Quarter Horse was referred to a Veterinary Hospital with episodes of abdominal discomfort, fever,anorexia, tachycardia, and tachypnea. The animal reportedly demonstrated episodes of blackened and fetid diarrhea afterthe ingestion of hay. A treatment was established upon arrival at the veterinary hospital, but the mare died after 12 hoursof monitoring. An autopsy examination performed soon after death revealed severe hyperemia of the mucosa of the cecum and colon, with multifocal cecal erosions and ulcerations. The principal histological lesion observed was necrotizingenterocolitis. Additional significant histopathologic lesions included widespread lymphoid depletion affecting the spleen,tonsils, and lymph nodes. An IHC assay designed to identify the antigens of N. helminthoeca (NH) in formalin fixed paraffin embedded (FFPE) tissues, identified antigens of intralesional neorickettsial organisms within macrophages of themucosa of the colon. Additionally, a PCR assay designed to amplify the 16S rRNA gene of Neorickettsia, amplified thedesired amplicon, but sequencing was frustrating.Discussion: A putative diagnosis of equine neorickettsiosis was established due to the combination of epidemiologicalevidence, pathologic findings, immunohistochemical identification of intralesional antigens of neorickettsial agents, andamplification...(AU)

Neonatal necrotizing enterocolitis rat model attenuated by a remote ischemic preconditioning in the pregnant

Purpose: To evaluate the effect of remote ischemic preconditioning (r-IPC) administered to pregnant rats, in the ileum of newborn rats subjected to hypoxia and reoxygenation. Methods: We used three pregnant rats and their newborn rats distributed in three groups: 1) Control (C) - Newborn rats born from a pregnant rat which did not undergo any intervention; 2) Hypoxia-Reoxygenation (H/R) - Newborn rats born from a pregnant rat which did not undergo any intervention, and were subjected to hypoxia-reoxygenation; 3) Remote Ischemic Preconditioning (r-IPC) - newborn rats born from a pregnant rat which was subjected to remote ischemic preconditioning twenty-four hours before giving birth and the newborn rats were subjected to hypoxia-reoxygenation. Segments of ileum were prepared for histological analysis by HE and immunohistochemistry by the Ki67 to evaluate cell proliferation, crypt depth and villus height and evaluation of apoptosis by cleaved caspase-3. Results: The intensity of the lesions was lower in the r-IPC than in the H/R group, showing significant difference (p 0.01). The r-IPC group showed a higher proliferative activity compared to the H/R group (p 0.01), with deeper crypts (r-IPC > H/R - p 0.05), and higher villi, showing significant difference (r-IPC > H/R - (p 0.01). The occurrence of apoptosis in the H/R group was lower in comparison to groups C and r-IPC, with significant difference (H/R r-IPC; p 0.05). Conclusion: The remote ischemic preconditioning applied to the pregnant rat protected the ileum of newborn rats subjected to hypoxia and reoxygenation, with decreased intensity of the lesions in the ileum mucosa and preservation of proliferative activity, keeping the villus height and crypt depth similar to group C.(AU)

Isolation and genotyping of Clostridium perfringens and Clostridium difficile in Capuchin Monkeys (Sapajus spp) / Isolamento e genotipagem de Clostridium perfringens and Clostridium difficile em macacos-prego (Sapajus spp)

The importance of Clostridium perfringens and C. difficile for most wild animal species remains unclear. This study aimed to isolate and genotype C. perfringens and C. difficile in stool samples from free-living and captive capuchin monkeys (Sapajus flavius and Sapajus libidinosus) in Brazil. Ten free-living S. flavius and 14 captive S. libidinosus were sampled for this study. To isolate C. difficile, stool samples were inoculated on plates containing cycloserine-cefoxitin fructose agar supplemented with horse blood and sodium taurocholate. Two different protocols for C. perfringens isolation were tested: direct plating onto selective agar and enrichment in brain heart infusion (BHI) broth followed by plating onto selective agar. C. difficile was not detected in the present study. The results were identical for both protocols tested for isolation of C. perfringens. Four samples (16.7%) were positive for C. perfringens type A, including one sample from a free-living animal (4.2%) and three from captive animals (12.5%), meaning there was no significant difference between these two groups. C. perfringens isolates were negative for all additional virulence factors evaluated, including enterotoxin encoding-gene (cpe) and beta-2 encoding-gene (cpb2). These results suggested that C. perfringens type A is found in the microbiota of capuchin monkeys, although it is less frequent than previously reported in domestic animals.

A importância de Clostridium perfringens e C. difficile para a maioria das espécies silvestres ainda não está clara. O objetivo do presente estudo foi isolar e genotipar C. perfringens e C. difficile em amostras de fezes de macacos-prego (Sapajus flavius e Sapajus libidinosus) de vida livre e criados em cativeiros no Brasil. Dez S. flavius de vida livre e 14 S. libidinosus de cativeiro foram incluídos no presente estudo. Para isolamento de C. difficile, as amostras de fezes foram inoculadas em agar cicloserina-cefoxitina frutose, suplementado com sangue e taurocolato de sódico. Para isolamento de C. perfringens, foram testados dois protocolos: plaqueamento direto em ágar seletivo e enriquecimento em caldo seguido de plaqueamento em ágar seletivo. C difficile não foi detectado no presente estudo. Os resultados foram idênticos para ambos os protocolos testados para isolamento de C. perfringens, resultando em quatro animais (16,7%) positivos para C. perfringens tipo A. Destes, uma amostra era de um animal de vida livre (4,2%) e três de animais de cativeiro (12,5%), não havendo diferença entre esses dois grupos. Os isolados de C. perfringens foram negativos para todos os fatores de virulência adicionais avaliados, incluindo o gene codificador de enterotoxina (cpe) e o gene codificador beta-2 (cpb2). O presente estudo sugere C. perfringens tipo A como parte da microbiota de macacos-prego, embora esse agente seja menos frequente como comensal, do que relatado anteriormente, em animais domésticos.

Isolation and genotyping of Clostridium perfringens and Clostridium difficile in Capuchin Monkeys (Sapajus spp) / Isolamento e genotipagem de Clostridium perfringens and Clostridium difficile em macacos-prego (Sapajus spp)

The importance of Clostridium perfringens and C. difficile for most wild animal species remains unclear. This study aimed to isolate and genotype C. perfringens and C. difficile in stool samples from free-living and captive capuchin monkeys (Sapajus flavius and Sapajus libidinosus) in Brazil. Ten free-living S. flavius and 14 captive S. libidinosus were sampled for this study. To isolate C. difficile, stool samples were inoculated on plates containing cycloserine-cefoxitin fructose agar supplemented with horse blood and sodium taurocholate. Two different protocols for C. perfringens isolation were tested: direct plating onto selective agar and enrichment in brain heart infusion (BHI) broth followed by plating onto selective agar. C. difficile was not detected in the present study. The results were identical for both protocols tested for isolation of C. perfringens. Four samples (16.7%) were positive for C. perfringens type A, including one sample from a free-living animal (4.2%) and three from captive animals (12.5%), meaning there was no significant difference between these two groups. C. perfringens isolates were negative for all additional virulence factors evaluated, including enterotoxin encoding-gene (cpe) and beta-2 encoding-gene (cpb2). These results suggested that C. perfringens type A is found in the microbiota of capuchin monkeys, although it is less frequent than previously reported in domestic animals.(AU)

A importância de Clostridium perfringens e C. difficile para a maioria das espécies silvestres ainda não está clara. O objetivo do presente estudo foi isolar e genotipar C. perfringens e C. difficile em amostras de fezes de macacos-prego (Sapajus flavius e Sapajus libidinosus) de vida livre e criados em cativeiros no Brasil. Dez S. flavius de vida livre e 14 S. libidinosus de cativeiro foram incluídos no presente estudo. Para isolamento de C. difficile, as amostras de fezes foram inoculadas em agar cicloserina-cefoxitina frutose, suplementado com sangue e taurocolato de sódico. Para isolamento de C. perfringens, foram testados dois protocolos: plaqueamento direto em ágar seletivo e enriquecimento em caldo seguido de plaqueamento em ágar seletivo. C difficile não foi detectado no presente estudo. Os resultados foram idênticos para ambos os protocolos testados para isolamento de C. perfringens, resultando em quatro animais (16,7%) positivos para C. perfringens tipo A. Destes, uma amostra era de um animal de vida livre (4,2%) e três de animais de cativeiro (12,5%), não havendo diferença entre esses dois grupos. Os isolados de C. perfringens foram negativos para todos os fatores de virulência adicionais avaliados, incluindo o gene codificador de enterotoxina (cpe) e o gene codificador beta-2 (cpb2). O presente estudo sugere C. perfringens tipo A como parte da microbiota de macacos-prego, embora esse agente seja menos frequente como comensal, do que relatado anteriormente, em animais domésticos.(AU)

Avaliação da eficiência dos toxóides recombinantes e de Clostridium perfringens na imunização de equinos

Clostridium perfringens tem sido classificado como principal agente patogênico gastrointestinal em potros com até 10 dias de idade, apesar dos quadros de enterocolites serem mais comuns em neonatos, animais jovens e adultos também podem ser acometidos. O quadro clínico que se manifesta na forma de cólica, diarreia sanguinolenta e evolui rapidamente para choque circulatório, é causado por ação da toxina (CPB) de C. perfringens tipo C. Esse agente bacteriano também está associado a graves quadros de mionecrose, geralmente fatais, por envolvimento da toxina (CPA) de C. perfringens tipo A. Para equinocultura, a importância dessas enfermidades deve-se a elevada mortalidade e a inexistência de vacinas comerciais, que garantam a imunização, principal forma de prevenção. Assim o objetivo deste trabalho foi ser o pioneiro em utilizar e avaliar a longevidade da resposta imune humoral no período de um ano, em equinos imunizados com diferentes concentrações (100, 200 e 400µg) de toxóides recombinantes CPA e CPB de C. perfringens tipos A e C, respectivamente, bem como comparar aos resultados obtidos em animais inoculados com toxóide comercial. Foram utilizados 50 animais da espécie equina, raça Mangalarga Marchador, de ambos os sexos, a partir de um ano de idade, sem histórico vacinal contra clostridioses. Os animais foram divididos aleatoriamente em cinco grupos de dez equinos: Grupo Vacina Recombinante 100µg (G1), Grupo Vacina Recombinante 200µg (G2), Grupo Vacina Recombinante 400µg (G3), Grupo Vacina Comercial (G4) e Grupo Controle Negativo (G5). Os equinos do G1, G2 e G3 foram vacinados com a vacina recombinante contendo diferentes concentrações dos toxóides recombinantes CPA e CPB 100, 200 e 400µg, respectivamente, G4 com vacina comercial e o G5 recebeu solução salina estéril (NaCl 0,9%). Todos os animais receberam duas doses de 2ml, por via intramuscular, na tábua do pescoço, nos dias zero e 28 após a primeira dose. As amostras de soro sanguíneo foram coletadas nos dias zero, 28, 56, 90, 120, 150, 180, 210, 240, 270, 300, 330 e 360 após a primeira vacinação. Os soros obtidos, após centrifugação foram submetidos a técnica de soroneutralização em camundongos. O teste de potência realizado no dia 56, demonstrou que as formulações de 200 e 400µg foram capazes de induzir resposta imune em todos os equinos inoculados, de acordo com os níveis exigidos na legislação, assim como, ao avaliar a longevidade (teste de eficiência) da resposta imune vacinal, as mesmas concentrações apresentaram níveis de anticorpos detectáveis até o dia 180, não havendo diferenças significativas entre os resultados obtidos. A vacina recombinante nas concentrações acima de 200µg foi capaz de estimular resposta imune humoral satisfatória em equinos.

Clostridium perfringens has been rated as the main gastrointestinal pathogen in foals up to age 10 days old, although enterocolitis is more common in neonates, young and adult animals may also be affected. The clinical picture which manifests itself in a form of colic, bloody diarrhoea and progresses rapidly towards a circulatory shock, is caused by the action of toxin (CPB) of C. perfringens type C. This bacterial agent is also associated with severe myonecrosis, usually fatal, due to the involvement of toxin (CPA) of C. perfringens type A. For equinoculture, the importance of these diseases is due to the high mortality and the absence of commercial vaccines, which ensure immunization, the main form to prevent. Therefore, the aim of the present work was to be pioneer in using and to analyse the longevity of humoral immune response in the period of one year, in horses immunized with different concentrations (100, 200 and 400µg) of recombinant toxoids CPA and CPB of C. perfringens types A and C, respectively, as well as compare to results acquired from inoculated animals with commercial toxoid. Fifty horses were used, Mangalarga Marchador breed, of both sexes, from the age of one year and up, no vaccine history against clostridiosis. The animals were randomly divided into five groups of ten horses: Recombinant Vaccine Group 100µg (G1), Recombinant Vaccine Group 200µg (G2), Recombinant Vaccine Group 400µg (G3), Commercial Vaccine Group (G4) and Negative Control Group (G5). G1, G2 and G3 horses have been vaccinated with the recombinant vaccine containing different concentrations of recombinant toxoids CPA and CPB 100, 200 and 400µg, respectively, G4 with commercial vaccine and G5 received sterile saline (NaCl 0.9%). All animals received two doses of 2ml, intramuscularly, in the neck, on days zero and 28 after the first dose. Blood serum samples were collected on days zero, 28, 56, 90, 120, 150, 180, 210, 240, 270, 300, 330 and 360 after the first vaccination. The serum obtained after centrifugation was submitted to the serum neutralization technique in mice. The potency test held on day 56 has demonstrated that the formulations of 200 and 400µg were able to induce an immune response in all inoculated horses, according to levels required by legislation, as well as, when assessing the longevity (efficiency test) of the immune response, the same concentrations presented levels of detectable antibodies until day 180, without significant differences between the results obtained. The recombinant vaccine in above concentrations 200 µg was able to stimulate a satisfactory humoral immune response in horses.

Effects of maternal ischemic preconditioning in the colon of newborn rats submitted to hypoxia-reoxygenation insult

PURPOSE: To evaluate the effects of maternal remote ischemic preconditioning (IPCr) in the colonic mucosa of newborn rats subjected to hypoxia and reoxygenation. METHODS: Newborn Wistar rats were divided into three groups. Control Group (CG), Hypoxia and Reoxygenation Group (HRG) and Remote Ischemic Preconditioning Group (IPCrG). Hypoxia and reoxygenation was performed 2x per day, with an interval of 6 hours, on the 1st, 2nd and 3rd days of life, with 10 minutes of CO2 at 100%, followed by 10 minutes O2 at 100%(HRG/IPCrG). The maternal IPCr was performed 24 hours before delivery by applying a rubber band tourniquet to the left hind limb (IPCrG). Segments of the colon underwent histological (HE) and immunohistochemical analysis for caspase-3 and COX - 2. RESULTS: The histological findings showed no intestinal mucosal damage in the CG group and severe lesions in HRG that was attenuated in the IPCrG (p<0.05). The expression of the apoptotic cells was lower in the HRG group than in the CG and IPCrG. The COX-2 expression was intense in HRG and attenuated in the IPCrG (p<0.05). CONCLUSIONS: Maternal IPCr protected the colonic mucosa of newborn rats subjected to hypoxia and reoxygenation, reducing the morphological alterations and inflammatory response. It ameliorates the occurrence of apoptosis, keeping the physiological process of renewal and regeneration in the epithelial lining of the colonic mucosa. (AU)

L-FABP and I-FABP expression in newborn rats changes inversely in the model of necrotizing enterocolitis

To determine the expression of hepatic L-FABP and intestinal I-FABP in an experimental model of necrotizing enterocolitis (NEC) in neonatal rats. Newborn Sprague-Dawley rats were divided into four groups: Control (C1) - exclusive breastfeeding at the first and sixth procedures (C6), NEC1 - fed formula milk and submitted to hypoxia and hypothermia at the first and sixth procedures (NEC6). The newborn pups were fed twice a day for three days, for a total of six procedures. Samples were collected for morphometric evaluation (body weight, liver weight, liver weight/body weight ratio, intestinal weight and intestinal/body weight ratio) and for immunohistochemical and Western blotting analysis. The values obtained were analyzed statistically, with the level of significance set at p<0.05. Morphometric measurements showed reduction of body and liver weights in the NEC group (p<0.05). Both immunohistochemistry and western blotting revealed that L-FABP expression in the liver was decreased and I-FABP expression in the ileum was increased in the NEC group (p<0.05). CONCLUSION: L-FABP and I-FABP expression changed inversely in the rat NEC model. These findings can contribute to a better diagnosis of NEC in human newborns.(AU)

Validation of protocol of experimental necrotizing enterocolitis in rats and the pitfalls during the procedure / Validação do modelo de enterocolite necrotizante experimental em ratos e as armadilhas durante sua execução

PURPOSE: To describe the difficulties of implementing the protocol of experimental necrotizing enterocolitis (NEC) in order to obtain a larger number of newborns affected with the disease and a lower mortality. METHODS: Term Sprague-Dawley newborns rats (22 days) were divided into four groups of 12 fetuses each (n = 48): EC - breastfed newborns; IH - breastfed newborns and subjected to a stress protocol by ischemia and hypothermia; ESB - formula-fed newborns (Esbilac®, PetAg, Hampshire, IL, USA) and NEC - formula-fed newborns and subjected to stress protocol. The parameters set for the study protocol were: milk concentration (0.19 g ml or 0.34 g/ml), diet instilled volume (according to body weight - 200 kcal/day/Kg - or progressive, according to acceptance), weight (gain, loss or maintenance) and duration of the experiment (72 hours or 96 hours). Data of body weight (BW), intestinal weight (IW) and the IW/BW ratio were obtained. Samples of terminal ileum were collected and analyzed by the degree of injury to the intestinal wall. Statistically significance was set to p<0.05. RESULTS: The established protocol with less mortality and increased number of NEC was with Esbilac® at a concentration of 0.19 g/ml of diet instilled volume of 0.1 ml, every 3 hours, for 72 hours. All infants fed with artificial milk lost weight. In the degree score of intestinal injury, the ESB, IH and NEC groups were considered positive for NEC with greater histological injury in the latter. CONCLUSION: The described NEC protocol in rats allowed a greater survival of puppies with a greater number of animals affected by the disease.(AU)

OBJETIVO: Relatar as dificuldades da execução do protocolo de enterocolite necrosante (ECN) experimental a fim de obter um maior número de neonatos comprometidos com a doença e menor mortalidade. MÉTODOS: Neonatos de ratas Sprague-Dawley nascidos a termo (22 dias) foram divididos em 4 grupos de 12 fetos cada (n=48): EC - neonatos amamentados pela mãe; IH - neonatos amamentados pela mãe e submetidos a estresse por isquemia e hipotermia, ESB - neonatos alimentados por leite artificial (Esbilac®, PetAg, Hampshire, IL, USA) e NEC - neonatos alimentados com fórmula e submetidos a protocolo de estresse. Os parâmetros estabelecidos para o protocolo de estudo foram: concentração do leite (0,19 g/ml ou 0,34 g/ml), volume de dieta instilada (de acordo com ganho de peso - 200 kcal/dia/kg - ou progressivo, de acordo com aceitação), peso (ganho, perda ou manutenção) e duração do experimento (72 h ou 96 h). Dados de peso corporal (BW), peso intestinal (IW) e a relação IW/BW foram obtidos. Amostras de íleo terminal foram coletadas e analisadas pelo grau de lesão da parede intestinal. Os dados foram analisados estatisticamente com p <0,05. RESULTADOS: O protocolo estabelecido com menor mortalidade e maior número de ECN foi com Esbilac® na concentração de 0,19 g/ml, volume de dieta instilada de 0,1ml, a cada 3 horas, durante 72 horas. Todos os neonatos alimentados com leite artificial perderam peso. Na escala do grau de lesão, os grupos ESB, IH e NEC foram considerados positivos para NEC com maior lesão histológica no último. CONCLUSÃO: O protocolo de NEC experimental em ratos estabelecido possibilitou uma maior sobrevivência dos neonatos com o maior numero de animais acometidos pela doença.(AU)

Doenças infecciosas em suínos nas fases de crescimento e terminação na região Sul do Brasil

A suinocultura brasileira representa uma importante atividade econômica para o país, o qual ocupa lugar de destaque na produção e exportação de carne suína no mundo. O destaque do país na produção de suínos, deve-se a melhorias na sanidade, manejo, produção integrada e, principalmente, no aprimoramento gerencial dos produtores. O primeiro artigo consistiu em determinar a frequência e a distribuição das doenças infecciosas (DI) diagnosticadas através de exame de necropsia e histopatologia em suínos nas fases de crescimento e terminação ao longo de 12 anos (2005-2016) no sul do Brasil. Foram avaliados 1906 laudos anatomopatológicos de suínos nas fases de crescimento/terminação, dos quais as DI corresponderam a 75,6% (1441 casos) do total. As infecções por circovírus suíno tipo 2 (PCV2) foram as mais frequentes, contabilizando 51,3% (739/1441) dos casos, seguidas por DI que afetam o sistema respiratório (30,1% dos casos). Dentre essas, destacam-se a influenza suína A (15,1%; 218/1441) e pneumonias bacterianas (15%; 216/1441). O diagnóstico de influenza exibiu uma frequência elevada entre os anos de 2010 a 2013, totalizando 43,1% (167/387) dos casos. Após este período, ambas DI respiratórias exibiram caráter endêmico. As DI que afetam o sistema digestório totalizaram 10,5% (151/1441) dos diagnósticos, com as seguintes condições: enterocolite por Salmonella spp. (43,7%; 66/151), enteropatia proliferativa por Lawsonia spp. (41,7%; 63/151) e colite por Brachyspira spp. (14,6%; 22/151). Além dessas, as polisserosites e meningites bacterianas representaram 5,8% (84/1441) e 2,3% (33/1441) dos casos diagnosticados, respectivamente. O segundo artigo descreve três surtos de doença por circovírus suíno tipo-2 (PCVD) com lesões envolvendo musculatura esquelética. Em um curso clínico de 7 a 10 dias, 92 suínos apresentaram apatia, emagrecimento e diarreia. Ainda, cerca de 30 dos suínos afetados, apresentavam dificuldade de locomoção, fraqueza muscular, paresia de membros pélvicos e decúbito permanente. Quatro suínos exibiram palidez em músculos esqueléticos dos membros pélvicos, torácicos e dorso-lombares. As lesões microscópicas observadas consistiam de miosite necrótica granulomatosa, predominantemente, em membros pélvicos e torácicos, e em menor intensidade nos músculos dorso-lombares. No exame imuno-histoquímico para PCV2 observou-se marcação multifocal acentuada, predominantemente, no citoplasma e núcleos de macrófagos, linfócitos e células gigantes multinucleadas, além de marcação discreta no citoplasma no citoplasma de fibras necróticas da musculatura esquelética. As amostras de músculo esquelético foram positivas na reação em cadeia de polimerase para PCV2 e a ampliação exibiu 99% de identidade com sequências pertencentes ao genótipo PCV2b.

Brazilian pig farms represent an important economic activity for the country, which occupies a prominent place in the production and export of pork in the world. The country's prominence in pork production is due to improvements in sanitation, management, integrated production and, mainly, in the managerial improvement of the producers. The first study aimed to determine the frequency and distribution of infectious diseases (ID) diagnosed through necropsy and histopathology examination in growing-finishing swine along 12 years (2005-2016) in Southern Brazil. A total of 1906 anatomopathological exams performed in growing-finishing swine were evaluated, of which ID accounted for 75.6% (1441 cases) of the total. Porcine circovirus type 2 (PCV2) infections were the most frequent, accounting for 51.3% of the cases (739/1441), followed by respiratory system ID (30.1% of the cases). Among these, the main conditions were swine influenza (15.1%; 218/1441) and bacterial pneumonia (15%; 216/1441). Influenza diagnosis had a higher frequency between 2010 and 2013, accounting for 43,1% (167/387) of the cases. After this period, both respiratory ID had an endemic occurrence. Digestive system ID accounted for 10.5% (151/1441) of the diagnosis, with the main conditions diagnosed: Salmonella spp. enterocolitis (43.7%; 66/151), Lawsonia spp. proliferative enteropathy (41.7%; 63/151) and Brachyspira spp. colitis (14.6%; 22/151). Besides these, polyserositis and bacterial meningitis represented, respectively, 5.8% (84/1441) and 2.3% (33/1441) of the cases diagnosed. The second study describes three outbreak of porcine circovirus disease (PCVD) with the involvement of skeletal muscle. In a clinical course of 7 to 10 days, 92 pigs had apathy, weight loss and diarrhea. Approximately 30 of these 92 pigs had stiff gait, muscle weakness, hind limb paresis, and recumbency. 4 pigs necropsied presented pale discoloration from hind and thoracic limbs, as well from dorsal lumbar skeletal muscles. The microscopical lesions consisted of granulomatous necrotizing myositis mainly of hind and thoracic limbs, and mildly from dorsal lumbar muscles. Immunohistochemistry exam for PCV2 revealed marked multifocal intracytoplasmic and intranuclear staining predominantly in macrophages, lymphocytes and multinucleated giant cells, with a lower amount in the cytoplasm of necrotic fibers of the skeletal muscle. Affected muscle samples were polymerase chain reaction positive for PCV2 and the amplicon exhibited 99% identity with sequences belonging to the PCV2b genotype.

Necrotic enterocolitis in pigs naturally infected by porcine circovirus type 2

Samples of intestine with necrotic enteritis from 63 pigs naturally infected with porcine circovirus type 2 (PCV2) were studied. Colon was the main target of PCV2 associated necrotic enteritis in 60 cases. Immunohistological investigations were carried out to detect the presence of PCV2 in necrotic lesions and to identify the type of cells infected by the virus. Crypt epithelial cells had positive labelling for PCV2 in 17 cases. Depletion of goblet cells occurred in 10 cases. In 24 necrotic enteritis cases, co-infection of PCV2 and Salmonella was identified. An increased rate of apoptosis in the crypt epithelial cells of the large intestine from PCV2 of naturally infected pigs was observed. Immunohistochemical findings confirmed the presence of PCV2 within cells from necrotic intestinal tissue, suggesting that PCV2 may play a role in the development of those lesions. Diagnosis of necrotic enteritis associated with PCV2 should be based on the detection of PCV2 antigen or DNA in the necrotizing lesions. However, bacteriological examination should be performed to rule out the presence of bacterial agents, since co-infections are likely to occur in PCV2 affected pigs.

Foram selecionadas amostras intestinais com enterite necrótica de 63 suínos naturalmente infectados pelo circovírus suíno tipo 2 (PCV2). Enterite necrótica associada com PCV2 ocorreu principalmente no cólon, em 60 casos. Análise imuno-histoquímica foi realizada para identificar a presença de PCV2 em lesões necróticas e o tipo de células infectadas pelo vírus. Células epiteliais das criptas apresentaram marcação positiva para PCV2 em 17 casos. Depleção de células caliciformes ocorreu em 10 casos. Em 24 casos de enterite necrótica, observou-se co-infecção por PCV2 e Salmonella. Foi observado um aumento no índice de apoptose nas células das criptas do intestino grosso de suínos naturalmente infectados com PCV2. Os achados imuno-histoquímicos e histopatológicos sugerem que a infecção por PCV2 das células do tecido intestinal pode ocasionar enterite necrótica. O diagnóstico de enterite necrótica associada com PCV2 deve ser baseado na detecção do antígeno ou do DNA viral nas lesões necróticas. Contudo, análise bacteriológica deve ser realizada para descartar a presença de agente bacteriano, já que co-infecções são comuns.

Necrotic enterocolitis in pigs naturally infected by porcine circovirus type 2

Samples of intestine with necrotic enteritis from 63 pigs naturally infected with porcine circovirus type 2 (PCV2) were studied. Colon was the main target of PCV2 associated necrotic enteritis in 60 cases. Immunohistological investigations were carried out to detect the presence of PCV2 in necrotic lesions and to identify the type of cells infected by the virus. Crypt epithelial cells had positive labelling for PCV2 in 17 cases. Depletion of goblet cells occurred in 10 cases. In 24 necrotic enteritis cases, co-infection of PCV2 and Salmonella was identified. An increased rate of apoptosis in the crypt epithelial cells of the large intestine from PCV2 of naturally infected pigs was observed. Immunohistochemical findings confirmed the presence of PCV2 within cells from necrotic intestinal tissue, suggesting that PCV2 may play a role in the development of those lesions. Diagnosis of necrotic enteritis associated with PCV2 should be based on the detection of PCV2 antigen or DNA in the necrotizing lesions. However, bacteriological examination should be performed to rule out the presence of bacterial agents, since co-infections are likely to occur in PCV2 affected pigs.

Foram selecionadas amostras intestinais com enterite necrótica de 63 suínos naturalmente infectados pelo circovírus suíno tipo 2 (PCV2). Enterite necrótica associada com PCV2 ocorreu principalmente no cólon, em 60 casos. Análise imuno-histoquímica foi realizada para identificar a presença de PCV2 em lesões necróticas e o tipo de células infectadas pelo vírus. Células epiteliais das criptas apresentaram marcação positiva para PCV2 em 17 casos. Depleção de células caliciformes ocorreu em 10 casos. Em 24 casos de enterite necrótica, observou-se co-infecção por PCV2 e Salmonella. Foi observado um aumento no índice de apoptose nas células das criptas do intestino grosso de suínos naturalmente infectados com PCV2. Os achados imuno-histoquímicos e histopatológicos sugerem que a infecção por PCV2 das células do tecido intestinal pode ocasionar enterite necrótica. O diagnóstico de enterite necrótica associada com PCV2 deve ser baseado na detecção do antígeno ou do DNA viral nas lesões necróticas. Contudo, análise bacteriológica deve ser realizada para descartar a presença de agente bacteriano, já que co-infecções são comuns.

Necrotic enterocolitis in pigs naturally infected by porcine circovirus type 2

Samples of intestine with necrotic enteritis from 63 pigs naturally infected with porcine circovirus type 2 (PCV2) were studied. Colon was the main target of PCV2 associated necrotic enteritis in 60 cases. Immunohistological investigations were carried out to detect the presence of PCV2 in necrotic lesions and to identify the type of cells infected by the virus. Crypt epithelial cells had positive labelling for PCV2 in 17 cases. Depletion of goblet cells occurred in 10 cases. In 24 necrotic enteritis cases, co-infection of PCV2 and Salmonella was identified. An increased rate of apoptosis in the crypt epithelial cells of the large intestine from PCV2 of naturally infected pigs was observed. Immunohistochemical findings confirmed the presence of PCV2 within cells from necrotic intestinal tissue, suggesting that PCV2 may play a role in the development of those lesions. Diagnosis of necrotic enteritis associated with PCV2 should be based on the detection of PCV2 antigen or DNA in the necrotizing lesions. However, bacteriological examination should be performed to rule out the presence of bacterial agents, since co-infections are likely to occur in PCV2 affected pigs.

Foram selecionadas amostras intestinais com enterite necrótica de 63 suínos naturalmente infectados pelo circovírus suíno tipo 2 (PCV2). Enterite necrótica associada com PCV2 ocorreu principalmente no cólon, em 60 casos. Análise imuno-histoquímica foi realizada para identificar a presença de PCV2 em lesões necróticas e o tipo de células infectadas pelo vírus. Células epiteliais das criptas apresentaram marcação positiva para PCV2 em 17 casos. Depleção de células caliciformes ocorreu em 10 casos. Em 24 casos de enterite necrótica, observou-se co-infecção por PCV2 e Salmonella. Foi observado um aumento no índice de apoptose nas células das criptas do intestino grosso de suínos naturalmente infectados com PCV2. Os achados imuno-histoquímicos e histopatológicos sugerem que a infecção por PCV2 das células do tecido intestinal pode ocasionar enterite necrótica. O diagnóstico de enterite necrótica associada com PCV2 deve ser baseado na detecção do antígeno ou do DNA viral nas lesões necróticas. Contudo, análise bacteriológica deve ser realizada para descartar a presença de agente bacteriano, já que co-infecções são comuns.

Avaliação de um modelo experimental de enterocolite necrosante neonatal em ratos / Evaluation of an experimental model of necrotizing enterocolitis in rats

OBJETIVO: Avaliar um modelo experimental de enterocolite necrosante em ratos proposto por Okur e colaboradores em 1995. MÉTODOS: Utilizou-se 28 ratos da raça EPM-Wistar no primeiro dia de vida, com peso entre 4 a 6 gramas. Os animais foram submetidos a hipóxia (H) colocando os filhotes em uma câmara de gás CO2 para sacrifício de roedores onde receberam um fluxo de ar contendo 100 por cento de CO2 durante 5 minutos. Após a hipóxia os animais foram reanimados (R) com fluxo de ar contendo O2 a 100 por cento, também durante 5 minutos. Os animais divididos em dois grupos: G1: controle (n=12): ratos não submetidos a H-R; G2: (n=16): ratos submetidos a H-R. Segmentos de intestino delgado e cólon foram preparados para análise histológica. O restante do intestino foi utilizado para dosagem de malondialdeído tecidual. RESULTADOS: Dosagem de malondialdeído do G1 foi em média 1,05 (0,44-2,03) e do G2 foi em média 2,60 (0,59- 6,4) nmol MDA/mg proteína. O G2 teve média significativamente maior do que a do grupo controle (p<0,002). Foi encontrada diferença estatisticamente significante entre os grupos de estudo quanto à distribuição do grau de lesão onde o grupo G1 apresentou graus significantemente menores do que o grupo G2. CONCLUSÕES: O modelo mostrou que a hipóxia neonatal em ratos provoca lesões na parede intestinal.. Apesar das lesões histológicas discretas é um bom método para avaliação da liberação de radicais livres teciduais. (AU)

OBJECTIVE: To evaluate an experimental model of necrotizing enterocolitis in rats proposed by OKUR e col. in 1995.METHODS: On their first day of life, 28 EPM-Wistar rats weighing between 4 and 6 grams were submitted to hypoxia (H) by placing them in a CO2 gas chamber for rodents' sacrifice, where they received a 100% CO2 air flow for 5 minutes. After the hypoxia the animals were reanimated (R) with a 100% O2 air flow, also for 5 minutes. The animals were allocated in two groups: G1: control (n=12): rats not submitted to H-R; G2: (n=16): rats submitted to H-R. Segments of the small intestine and colon were prepared for histological analysis. The remaining intestine was used to measure tissular malondialdehyde.RESULTS: Mean malondialdehyde dosages were 1.05 (0.44-2.03) and 2.60 (0.59- 6.4) nmol MDA/mg protein for G1 and G2, respectively. G2's mean value was significantly higher than in the control group (p<0.002). Significant statistical difference between the studied groups was found in relation the level of injury, with G1 presenting significantly lower levels than G2.CONCLUSIONS: The model showed that neonatal hypoxia may cause intestinal wall injury in rats. Despite the discreet histological injuries found, the method is suitable for evaluation of tissular free radicals.