The first study was conducted to evaluate the dietary inclusion of effective microorganisms (EM) on body weight (BW), feed intake, feed conversion ratio (FCR) and mortality of broilers, which had been either challenged or non-challenged with Clostridium perfringens (1mL of 1 x 108 CFU/mL orally). Six hundred and forty day-old Ross 788 broiler chicks were randomly allocated to thirty-two pens in groups of twenty birds per pen, giving a stocking density of ±18 birds per square meter from zero to forty days of age. The facility consisted of two rooms with sixteen pens per room. All the chicks were fed on a commercial maize-soya type diet, including a mash starter and a mash grower/finisher feed. At two weeks of age 320 chicks were inoculated with Clostridium perfringens type A through oral administration. The study had a randomised block design with four replicates and four treatments as: 1) Control-unmedicated; 2) antibiotic growth promoters (AGP) added to feed at 33g/kg; 3) EM added to feed and water at 50g/kg and 50mL/L respectively; and 4) AGP in feed at 33g/kg and EM (50g/kg) in feed and water (50mL/L). The inclusion of EM negatively affected water palatability that resulted in reduced water intake and increased FCR for the non-challenged broilers at 21 days of age. However, because feed intake was not affected, it was suggested that EM should rather be supplemented through the feed rather than through the water. The use of AGP alone or in combination with EM proved (P<0.05) broiler production performance. The cumulative feed intake, BW, FCR, average daily gain (ADG), cumulative water intake and production efficiency factor (PEF) of challenged broilers were not different (P>0.05) at 40 days of age. In this study the incidence of mortality was low (2.2%) and examination of livers and intestines showed only mild necrotic enteritis lesions. In conclusion, the findings of this study showed that EM under the current dosage failed to improve broiler production performance. A second experiment was conducted as a follow-up study to evaluate the effect of EM on broiler performance when supplemented through the feed only. Cloacal swabs were taken from all day-old chicks and a day after inoculation with Clostridium perfringens for laboratory analysis of the microorganisms in the gut. All the chicks were fed on a commercial two-phase maize-soya type diet consisting of a mash starter and a mash grower/finisher feed with additional fishmeal. The chicks from one room were inoculated orally with Clostridium perfringens, while the chicks from the other room remained unchallenged. The challenged group was inoculated orally with 1 mL (1 x 108 CFU/mL) of Clostridium perfringens as a single dose on day 14. The EM was supplemented to the broilers from day one through the feed. The supplementation of EM through the feed showed a poor performance for non-challenged whilst for the challenged showed an improved performance at 3 weeks. Both non-challenged and challenged broilers given EM had a poor performance at 6 weeks of age, and this showed inconsistent results throughout the experiment. However, it was found that the combination of both AGP and EM had a better performance than EM alone. It is noted when the results of broilers nonchallenged versus challenged control groups were compared, the challenged ones showed a better performance. The broilers showed a low mortality of 1.3% and the causes were not related to the incidence of necrotic enteritis. The gross examination of the broilers inoculated orally with Clostridium perfringens showed mild intestinal lesions. Copyright / Dissertation (MSc(Agric))--University of Pretoria, 2010. / Animal and Wildlife Sciences / unrestricted

A 67-years-old male presented with periumbilical abdominal pain, fever and jaundice. His anaerobic blood culture was positive for clostridium perfringens. Computed tomogram scan of the abdomen and abdominal ultrasound showed normal gallbladder and common bile duct (CBD). Subsequently magnetic resonance cholangiopancreaticogram showed choledocholithiasis. Endoscopic retrograde cholangiopancreaticogramwith sphincterotomy and CBD stone extraction was performed. The patient progressively improved with antibiotic therapy Choledocholithiasis should be considered as a source of clostridium perfringens bacteremia especially in the setting of elevated liver enzymes with cholestatic pattern.

The bacterium Clostridium perfringens is a gram-positive anaerobe responsible for many diseases in man and other animals, the most common of which is acute food poisoning (AFP). It is estimated that nearly 240,000 cases of AFP occur each year in the U.S. The C. perfringens spore plays an important role in this infection. The heat resistance of the spore allows the organism to survive the cooking process, grow in the cooling food, and infect the victim. Despite the occurrence of this disease and the importance of the spore to this process, little work has been performed to determine how heat resistance is obtained and maintained by C. perfringens spores. In this work we study the spores and sporulation process of C. perfringens to determine what factors are most important in the formation of a heat resistant spore. We analyzed the spores produced by nine wild-type strains, including five heat-resistant food poisoning isolates and four less heat-resistant environmental isolates. We determined that threshold core density and a high ratio of cortex peptidoglycan relative to germ cell wall were necessary components of a highly heat-resistant spore. In order to test these observations, we constructed two mutant strains. The first could not achieve the necessary level of core dehydration and rapidly lysed in solution. The second mutant had a reduced amount of cortex relative to germ cell wall, and suffered a corresponding decrease in heat resistance as compared to our wild-type strains. The mutant strains supported the observations drawn from our wild-type strains. Dipicolinic acid is a major component of bacterial spores and is necessary for spore heat resistance. The Cluster I clostridia, including C. perfringens, lack the known DPA synthase operon, spoVF. We developed an in vitro assay for detecting DPA synthetase activity and purified the active enzyme from sporulating C. perfringens crude extract and identified the proteins with mass spectrometry. These results identified the electron transfer flavoprotein alpha chain (EtfA) as the DPA synthase of C. perfringens. Inactivating the etfA gene in C. perfringens resulted in a strain that could begin, but not complete, the sporulation process and produced dramatically lower amounts of DPA than the wild-type. The purified enzyme was shown to produce DPA in vitro and utilized FAD as a preferred cofactor. The results of this research may lead to future techniques to decrease the occurrence of the diseases caused by C. perfringens spores and treatments which may carry over to the diseases caused by similar organisms. / Ph. D.

Clostridium perfringens é um dos microrganismos mais freqüentemente envolvidos em surtos de enfermidades transmitidas por alimentos. Este microrganismo pode ser classificado em cinco tipos toxigênicos (A-E), de acordo com a detecção dos genes codificadores de suas principais toxinas: alfa (cpa), beta (cpb), épsilon (etx) e iota (iap), sendo que técnicas moleculares empregando a PCR são atualmente utilizadas para genotipagem desses isolados. Alguns isolados de C. perfringens produzem uma enterotoxina (CPE) que é responsável pelos sintomas clínicos desenvolvidos em casos de toxinfecção alimentar, sendo que esta toxina é codificada pelo gene cpe. A simples detecção de C. perfringens em um alimento, mesmo naqueles suspeitos de causar surtos, não é suficiente para considerá-lo como de risco à saúde do consumidor. Isto porque dentre os isolados de C. perfringens apenas um número muito pequeno apresenta o gene cpe. Além disso, isolados de C. perfringens não produtores de CPE estão amplamente disseminados no ambiente, em alimentos e mesmo em fezes de pessoas. Desta forma, com o presente estudo verificou-se a freqüência de C. perfringens dentre isolados de clostrídios sulfito redutores, a freqüência de C. perfringens potencialmente enterotoxigênicos e sua variabilidade genética, de modo a evidenciar a importância dessas cepas como causadoras de doenças, além de fornecer subsídios para melhorar os conhecimentos sobre as características das cepas circulantes em nosso meio. Foram utilizados 335 isolados de clostrídios sulfito redutores provenientes de alimentos (126), solo (84) e fezes de animais (125). Dos 335 isolados, 146 (43,6%) foram caracterizados, através de reações bioquímicas e moleculares, comoC. perfringens, sendo 75 isolados (59,5%) provenientes de alimentos, 43 (51,2%) de solo e 28 (22,4%) de fezes de animais. Todas as cepas de C. perfringens analisadas foram tipadas como C. perfringens tipo A. Dos 75 isolados de C. perfringens provenientes de alimentos, 20 apresentaram o gene cpe, sendo 13 (65%) com localização cromossomal; nas demais cepas não foi possível determinar sua localização. Nos isolados de C. perfringens provenientes de solo e das fezes de animais não se verificou a presença desse gene. Das 20 cepas de C. perfringens que apresentaram o gene cpe detectou-se em 15 a produção de enterotoxina; as cinco cepas restantes não apresentaram esporulação no meio DUNCAN STRONG modificado, não sendo possível avaliar sua atividade enterotoxigênica. As 146 cepas de C. perfringens quando submetidas à PFGE geraram 69 perfis PFGE distintos, sendo 42 exclusivos para uma única cepa, indicando uma grande variabilidade genética, entre isolados provenientes de amostra de alimentos, fezes ou solo. A utilização de clostrídios sulfito redutores, ou mesmo de C. perfringens como indicador de possível risco à saúde dos consumidores pode levar à condenação desnecessária de alimentos, uma vez que existe baixa correlação entre costrídios sulfito redutores e C. perfringens, independente da fonte de isolamento, além da baixa freqüência do gene cpe nas cepas estudadas. / Clostridium perfringens is one of the most frequently microorganism involved in outbreaks of foodborne diseases. This microorganism can be classified into five toxigenic types (A to E), according to the detection of genes encoding its major toxins: alpha (cpa), beta (cpb), epsilon (etx) and iota (iap). Molecular techniques using PCR are currently used for genotyping this isolates. Besides the major toxins, some isolates of C. perfringens produce an enterotoxin (CPE) that is responsible for clinical symptoms developed in cases of food poisoning. This enterotoxin is encoded by the cpe gene. The simple detection of C. perfringens in food, even in those suspected of causing outbreaks, is not enough to consider it as a risk to consumers´ health. This happens because among the isolates ofC. perfringens only a very small number shows the cpe gene. In addition, isolates of C. perfringens that do not produce CPE are widespread in the environment, food and even in feces of humans. Thus, the present study examined the frequency of C. perfringens isolates among sulfite reducing clostridia, the frequency of potentially enterotoxigenic C. perfringens and its genetic variability in order to highlight the importance of these strains in causing diseases, and provides subsidies to improve the knowledge about the strains that are circulating in our environment. A total of 335 isolates of sulfite reducing clostridia from foods (126), soil (84) and animal feces (125) were used. Among the 335 isolates, 146 (43.6%) were characterized by biochemical and molecular reactions as C. perfringens, being 75 (59.5%) from foods, 43 (51.2%) from soil and 28 (22.4%) from animal feces. All strains of C. perfringens were typed as C. perfringens type A. Of the 75 isolates of C. perfringens from food, 20 had the cpe gene, and in 13 (65%) the gene was chromosomally located. In the other strains it was not possible to determine the location of this gene. In isolates of C. perfringens from soil and animal feces the cpe gene was not present. Amongst the 20 strains of C. perfringens positive for cpe, enterotoxin production was detected in 15. Five strains showed no sporulation in the medium modified Duncan Strong, being not possible to verify their enterotoxigenic activity. All C. perfringens were subjected to PFGE and generated 69 different PFGE profiles, being 42 unique to a single strain, indicating a great genetic variability among isolates from food, feces or soil. The use of sulfite reducing clostridia, or even C. perfringens as an indicator of possible health risk to consumers can lead to unnecessary condemnation of food, since there is low correlation between sulfite reducing clostridia and C. perfringens, regardless of source of isolation. This study also shows a low frequency of cpe gene in the strains indicating the low risk in causing foodborne disease.

O setor avícola apresenta o maior crescimento em volume produzido dentre todos os setores cárneos no Brasil. A grande participação dos produtos avícolas na alimentação humana somada ao risco do desenvolvimento de resistência bacteriana, levaram a União Européia (UE) a abolir utilização de antimicrobianos como aditivos e de forma profilática na ração de animais. A remoção dos aditivos associada ao sistema de criação intensivo acabaram por fazer que doenças, até então consideradas controladas, se tornassem reemergentes. A enterite necrótica aviária (NE) é considerada um exemplo. De forma geral, condições estressoras predispõem ao desenvolvimento de doenças, sendo o calor um dos estressores mais comuns que ocorrem em granjas aviárias. Este estudo enfoca o efeito do estresse térmico por calor (35±1ºC) sobre o desenvolvimento da NE em frangos de corte. Para isso, 60 frangos de corte machos foram divididos em 6 grupos experimentais: 1 Grupo Controle; 2 Grupo Controle Estressado (C/HS35); 3 Grupo Tioglicolato (T); 4 Grupo Tioglicolato Estressado (T/HS35); 5 Grupo Infectado (I); 6 Grupo Infectado Estressado (I/HS35). A infecção experimental com Clostridium perfringens foi feita por via oral, com a bacteria em meio de cultura misturada a ração, do 15º ao 21º dia de vida nos grupos I e I/HS35. O estresse por calor (35±1º C) foi realizado do 14º ao 21º dia de vida das aves dos grupos estressados. Durante todo período experimental os animais foram mantidos em isoladores. Em relação aos animais não estressados, os animais submetidos ao estresse por calor apresentaram: 1- menor escore lesional macro e microscópico no intestino delgado; 2- maior concentração de IgA no lavado intestinal do duodeno; 3 - menor concentração de IgA no jejuno; 4 - redução dos níveis séricos de IgA e IgY; 5 - maior concentração sérica de IgM; 6 - diminuição qualitativa evidente dos heterofilos intestinais em relação aos animais infectados e estressados. Portanto, mostrou-se que o estresse por calor apresentou efeito imunomodulador importante, ao reduzir a inflamação intestinal. Este achado associa-se provavelmente à diminuição da imunidade inata por redução da migração de heterófilos para a mucosa intestinal, desta forma prevenindo a manifestação de um um quadro clínico mais grave de NE, fato associado à diminuição das lesôes desencadeadas pelo processo inflamatório heterofílico. / The poultry sector presented the highest growth in the volume of production among all meat sectors in Brazil. The great participation of poultry products on human diet together with the risk of food and environmental contamination by resistant bacteria led the European Union (EU) countries to abolish the use of antibiotics as feed additives in animal production. This fact associated with the intensive farming system are being reported as responsible for the re-emergence of some already controlled diseases. The avian necrotic enteritis (NE) exemplify such an effect. Generally, stressful conditions are predisponent factors for disease development; heat stress is one of the most common stressor in poultry farms. This study focuses on the effects of heat stress (35 ± 1 º C) on the development of NE in broilers. For this purpose, 60 male broilers were divided into 6 groups: 1 - control group, 2 - stressed control group (C/HS35) 3 - thioglycolate group (T) 4 - thioglycolate stressed group (T/HS35); 5 - infected group (I) 6 - infected stressed group (I/HS35). Experimental infection with Clostridium perfringens, grown in thioglycollate broth medium, was given through the feed to the birds of groups I and I/HS35 from the 15th to 21st days of life. The heat stress (35 ± 1 °C) was induced continuously from the 14th to the 21st day of life in birds of the stressed groups. Throughout the experimental period the animals were kept in isolators. Compared to non-stressed animals, broilers subjected to heat stress showed: lower gross and microscopic score lesions in the small intestine; increased concentrations of IgA in duodenal lavage and decreased IgA concentrations in the jejunum; smaller concentrations of serum IgA and IgY; increased concentration of serum IgM; reduction in gut number of heterophils in the thioglycolate treated and in the infected groups. Therefore, this experimental model showed that heat stress presented a significant immunomodulatory role on the induced NE, most probably because it reduced intestinal inflammation via decrease in heterophils migration to the intestinal mucosa, which in turn might had reduced tissue damage during infamation, hence preventing the development of a more severe form of NE.

C. perfringens é uma bactéria anaeróbia presente no intestino delgado do homem e animais em equilíbrio e, sob a ação de alguns fatores predisponentes como mudança brusca de alimentação ou super alimentação, stress no manejo ou alto parasitismo intestinal, há a proliferação do microrganismo com a consequente produção de potentes toxinas que provocam a morte do animal. Dentre as toxinas principais destaca-se a toxina alfa, importante fator de virulência, produzida por todos os tipos de C. perfringens, sendo os pertencentes ao tipo A os maiores produtores. A fim de caracterizar o microrganismo em suspeitas de enterotoxemia em ruminantes, trabalhamos com 61 amostras de intestino delgado de bovinos e 12 de ovinos como grupo estudo e no grupo controle composto de animais hígidos levados ao abate, 73 amostras de intestino delgado de bovinos e 24 de ovinos. Foram realizados procedimentos de isolamento e tipagem molecular de C. perfringens e quantificação celular, detecção molecular da toxina &beta;2, além de avaliações moleculares qualitativa (PCR convencional) e quantitativa (PCR em tempo real) do gene da toxina alfa dos diferentes isolados. Em 29 amostras do grupo estudo bovino (47,54%) e em 4 (33,33%) do grupo estudo ovino isolou-se o microrganismo, em contrapartida no grupo controle bovino não houve isolamento do bacilo e 5 amostras do grupo controle ovino (20,83%) foram positivas. Houve diferença estatisticamente significante somente entre os grupos de bovinos (p<0,05). Todos os isolados (100%) foram classificados como tipo A, e os resultados das quantificações celulares de C. perfringens revelaram que todos os bovinos controle apresentaram <10 UFC/g de conteúdo enquanto que o grupo estudo apresentou mediana de 104 UFC/g com variações de <10 UFC/g até 108 UFC/g. Nos ovinos, a mediana no grupo controle foi 101 UFC/g assim como no grupo estudo, entretanto com clara separação de valores entre os grupos. Tanto na PCR convencional quanto na PCR em tempo real para detecção do RNAm da toxina alfa foi observado limiar de detecção de 102 cópias de cDNA por reação, porém provavelmente devido aos valores das amostras estarem próximos ao limite da sensibilidade analítica da reação, não foi observada boa reprodutibilidade da última. Já na reação molecular convencional, observou-se a presença de detecção de RNAm da toxina alfa em 60,52% dos isolados o que revela alguma diferença da presença do transcrito entre as culturas, já que nas cepas restantes não foi detectada a presença do RNAm em questão. A pesquisa do gene da toxina &beta;2 revelou sua presença em 54,55% dos isolados de C. perfringens corroborando com a afirmativa de que o gene está amplamente distribuído entre os ruminantes. A metodologia aplicada para avaliação da expressão do gene da toxina alfa nos isolados mostrou que há diferenças dos níveis de transcrição porém não permitiu quantificar esses valores. A tipagem molecular corrobora com outros estudos quanto à importância epidemiológica do tipo A nos quadros de enterotoxemia em ruminantes, e os dados da quantificação celular permite-nos concluir que animais sadios possuem um nível basal de C. perfringens <10 UFC/g de conteúdo que não possibilita o seu isolamento. / C. perfringens is an anaerobe present in small intestine of man and animals in equilibrium, and under some predisposing factors such as sudden feeding change or super feeding, rough management or high intestinal parasitism, the microorganism multiplies with the consequent production of potent toxins that can cause animal death. Amongst the main toxins, alpha toxin is an important virulence factor, that is produced by all C. perfringens types, and those belonging to type A are its higher producer. Aiming to characterize the microorganism in ruminants suspect of enterotoxaemia, we evaluated 61 bovine small intestinal samples and 12 sheep small intestines as the study group, and for the control group composed by higid animals led to slaughterhousing, 73 bovine small intestines and 24 ovine samples. We performed microbiological culture and molecular typing of C. perfringens isolates, cellular quantification, molecular detection of 2 toxin, and qualitative and quantitative molecular evaluations of alpha toxin from different isolates by means of conventional PCR and real time PCR, respectively. In 29 samples from the bovine study group (47.54%) and in 4 (33.33%) from ovine study group the microorganism was isolated, however in the bovine control group there was no isolation success and 5 samples from sheep control group (20.83%) were positive. There was statistically significant difference only between bovine groups (p<0,05). All isolates (100%) were classified as type A, and C. perfringens cellular quantification results showed that every control bovine presented <10 CFU/g of intestinal contents while the study group presented a median of 104 CFU/g with results ranging from <10 CFU/g to 108 CFU/g. In sheep, the median value in the control group was 101 CFU/g as in the study group, but with a clear division of values between the groups. We observed the threshold detection of 102 cDNA copies per reaction in both conventional and real time PCR reactions for alpha toxin mRNA detection, however since the samples quantification values were close to the analytical sensitivity of the test, we could not observe the reproducibility in the last technique. In the conventional PCR reaction, alpha toxin mRNA was detected in 60.52% of the isolates. This result reveals some difference in the transcript presence among the cultures, since we could not detect the presence of the described mRNA in the other isolates. Beta2 toxin gene was detected in 54.55% of C. perfringens isolates corroborating with the affirmative that this gene is widely distributed among ruminants. The methodology presented herein for the evaluation of alpha toxin gene expression showed that there are differences in the transcription levels, however it didnt allow to quantify these values. Molecular typing results agree with other studies regarding the epidemiological importance of type A in the enterotoxaemia processes in ruminants, and the cellular quantification data allow us to conclude that healthy animals show a basal level of C. perfringens <10 CFU/g of intestinal content that doesnt allow its isolation.

O setor avícola apresenta o maior crescimento em volume produzido dentre todos os setores cárneos no Brasil. A grande participação dos produtos avícolas na alimentação humana somada ao risco do desenvolvimento de resistência bacteriana, levaram a União Européia (UE) a abolir utilização de antimicrobianos como aditivos e de forma profilática na ração de animais. A remoção dos aditivos associada ao sistema de criação intensivo acabaram por fazer que doenças, até então consideradas controladas, se tornassem reemergentes. A enterite necrótica aviária (NE) é considerada um exemplo. De forma geral, condições estressoras predispõem ao desenvolvimento de doenças, sendo o calor um dos estressores mais comuns que ocorrem em granjas aviárias. Este estudo enfoca o efeito do estresse térmico por calor (35±1ºC) sobre o desenvolvimento da NE em frangos de corte. Para isso, 60 frangos de corte machos foram divididos em 6 grupos experimentais: 1 Grupo Controle; 2 Grupo Controle Estressado (C/HS35); 3 Grupo Tioglicolato (T); 4 Grupo Tioglicolato Estressado (T/HS35); 5 Grupo Infectado (I); 6 Grupo Infectado Estressado (I/HS35). A infecção experimental com Clostridium perfringens foi feita por via oral, com a bacteria em meio de cultura misturada a ração, do 15º ao 21º dia de vida nos grupos I e I/HS35. O estresse por calor (35±1º C) foi realizado do 14º ao 21º dia de vida das aves dos grupos estressados. Durante todo período experimental os animais foram mantidos em isoladores. Em relação aos animais não estressados, os animais submetidos ao estresse por calor apresentaram: 1- menor escore lesional macro e microscópico no intestino delgado; 2- maior concentração de IgA no lavado intestinal do duodeno; 3 - menor concentração de IgA no jejuno; 4 - redução dos níveis séricos de IgA e IgY; 5 - maior concentração sérica de IgM; 6 - diminuição qualitativa evidente dos heterofilos intestinais em relação aos animais infectados e estressados. Portanto, mostrou-se que o estresse por calor apresentou efeito imunomodulador importante, ao reduzir a inflamação intestinal. Este achado associa-se provavelmente à diminuição da imunidade inata por redução da migração de heterófilos para a mucosa intestinal, desta forma prevenindo a manifestação de um um quadro clínico mais grave de NE, fato associado à diminuição das lesôes desencadeadas pelo processo inflamatório heterofílico. / The poultry sector presented the highest growth in the volume of production among all meat sectors in Brazil. The great participation of poultry products on human diet together with the risk of food and environmental contamination by resistant bacteria led the European Union (EU) countries to abolish the use of antibiotics as feed additives in animal production. This fact associated with the intensive farming system are being reported as responsible for the re-emergence of some already controlled diseases. The avian necrotic enteritis (NE) exemplify such an effect. Generally, stressful conditions are predisponent factors for disease development; heat stress is one of the most common stressor in poultry farms. This study focuses on the effects of heat stress (35 ± 1 º C) on the development of NE in broilers. For this purpose, 60 male broilers were divided into 6 groups: 1 - control group, 2 - stressed control group (C/HS35) 3 - thioglycolate group (T) 4 - thioglycolate stressed group (T/HS35); 5 - infected group (I) 6 - infected stressed group (I/HS35). Experimental infection with Clostridium perfringens, grown in thioglycollate broth medium, was given through the feed to the birds of groups I and I/HS35 from the 15th to 21st days of life. The heat stress (35 ± 1 °C) was induced continuously from the 14th to the 21st day of life in birds of the stressed groups. Throughout the experimental period the animals were kept in isolators. Compared to non-stressed animals, broilers subjected to heat stress showed: lower gross and microscopic score lesions in the small intestine; increased concentrations of IgA in duodenal lavage and decreased IgA concentrations in the jejunum; smaller concentrations of serum IgA and IgY; increased concentration of serum IgM; reduction in gut number of heterophils in the thioglycolate treated and in the infected groups. Therefore, this experimental model showed that heat stress presented a significant immunomodulatory role on the induced NE, most probably because it reduced intestinal inflammation via decrease in heterophils migration to the intestinal mucosa, which in turn might had reduced tissue damage during infamation, hence preventing the development of a more severe form of NE.

Die nekrotische Enteritis (NE) und Kokzidiose des Huhnes sind häufige Darmerkrankungen weltweit und führen jedes Jahr zu hohen Wirtschaftsverlusten als Folge der Mortalität und Kosten für Behandlung und Bekämpfung. Die beiden Erkrankungen werden meistens bei Mastbroilern zwischen der 3. und 6. Lebenswoche festgestellt. Weil die leistungsfördernden Antibiotika im Geflügelfutter in der EU nicht mehr eingesetzt werden dürfen, ist das Risiko der NE in den letzten Jahren gestiegen. Fischmehl, Stress und Krankheiten sind prädisponierende Faktoren für die Entstehung der NE aber auch die Hühnerkokzidiose spielt in der Hinsicht eine wichtige Rolle. Die Mechanismen der Pathogenese bei der Wechselwirkung zwischen Kokzidiose und NE sind unklar. Bisher wurden verschiedene Infektionsmodelle zur Erforschung der NE unter Beteiligung von Ko-Infektionen zwischen Eimerien und C. perfringens eingesetzt. Dabei steht neben der Krankheitsentstehung auch die effiziente Bekämpfung dieser Erkrankung als großes Problem der Geflügelindustrie im Forschungsmittelpunkt. C. perfringens ist auch bei gesunden Tieren ein häufiger Darmbewohner, wobei bestimmte Stämme verschiedene Toxintypen wie Alpha-, TpeL- und Beta-Toxine produzieren können und damit ursächlich zur Entstehung der NE beitragen, aber ohne andere prädisponierende Auslöser kommt es in der Regel nicht zum Ausbruch. In der vorliegenden Arbeit wurden zwei In-vivo-Modelle zur experimentellen Induktion der NE des Huhnes entwickelt. Ziele waren sowohl die genauere Untersuchung der Pathogenese der NE durch Ko-Infektionen mit Eimerien und Kokzidien als auch die Evaluierung von potentiellen neuartigen Bekämpfungsmöglichkeiten. Außerdem wurde ein In-ovo-Modell etabliert.

A enterite necrótica causada por Clostridium perfringens é, comprovadamente, um grande problema para frangos de corte, seja sob a forma clínica ou subclínica , com elevados prejuízos produtivos. Em perus, não está claramente identificada a influência deste patógeno sobre os resultados produtivos, e provavelmente por razões econômicas, o volume de pesquisa nesta área é limitado. Em dois experimentos consecutivos, conduzidos em granjas experimentais diferentes, e sobre cama nova e reutilizada, perus de 14 dias, sem medicação com anticoccidianos, foram inoculados, a partir de uma amostra de campo patogênica, com uma superdosagem de aproximadamente 1 x 1010 UFC/ml de Clostridium perfringens, tipo A; bactéria anaeróbica Gram positiva, que compõe a microflora intestinal dos perus. Lesões necróticas severas, com grande destruição da mucosa intestinal, foram observadas após o 4º dia de inoculação. Nos dois experimentos, a resposta dos perus à infecção foi diferente da descrita em frangos de corte. A mortalidade foi muito baixa ou nula e a recuperação das aves, independente da medicação utilizada, foi rápida com pouco ou nenhum comprometimento zootécnico. Escores das lesões histológicas foram desenvolvidos, para tentar correlacionar as lesões macroscópicas com as lesões microscópicas, mas a correlação foi baixa. Frente ao alto desafio observado, não foi possível avaliar adequadamente a ação do antimicrobiano utilizado. As respostas frente aos desafios de Clostridium perfringens nos perus, aparentam ser diferentes em relação às observadas em frangos de corte, sendo necessária cautela ao extrapolar padrões de uma espécie para outra. O uso de critérios subjetivos nas avaliações podem comprometer a tomada de decisão em relação aos tratamentos e as respostas esperadas. / Necrotic enteritis caused by Clostridium perfringens represents a major challenge in broilers, causing clinical or sub clinical diseases, and results in important economic losses for the poultry industry. In turkeys, however, the importance of this pathogen is not clearly defined, and there are few studies assessing its effects on the performance of turkeys, most probably for economic reasons. In two consecutive trials, conducted in two experimental farms, 14 day old turkey poults were reared on new and reused wood shavings litter, without any anticoccidials in the feed, and were inoculated with an overdose of approximately 1x1010 CFU/ml of a pathogenic field sample of Clostridium perfringens type A. This Gram positive anaerobic bacterium is a normal inhabitant of the gut micro flora in the enteric tract of turkeys. Severe necrotic lesions, with major damage to the intestinal mucosa were observed on Day 4 after inoculation. In both trials, the response to the turkeys to the clostridial infection was clearly different from what is described in the literature for broilers. The mortality rate was very low or inexistent, and the birds in all treatments groups recovered very fast, with little or no impact on performance results. An attempt was made to establish a pattern to correlate histological lesion scores with macroscopic and microscopic lesions, but the resulting correlation was very low. Considering the high level of challenge induced in both trials, it was not possible to evaluate in activity of the antibiotic in proper terms. The response of turkeys to a high level of challenge with Clostridium perfringens seems to be different when compared to what is observed in broilers under similar experimental conditions, and caution should be applied when using the same diagnostic methods for different species. The use of subjective criteria to assess and describe the lesions can lead to erroneous treatments decisions and misinterpretation of the responses to treatment.

A diarréia é o principal evento clínico observado no período neonatal em leitões (como conseqüência das doenças entéricas). Além de contribuir para piora no ganho de peso diário e conversão alimentar do animal causa aumento na mortalidade e gastos com medicamentos. O presente trabalho teve como objetivo determinar a freqüência dos principais agentes virais (rotavírus), bacterianos (E. coli, Clostridium perfringens tipo A e C e Clostridium difficile) e parasitários (coccídeos e Cryptosporidium spp.) envolvidos na diarréia neonatal em leitegadas caso (com diarréia) e controle (sem diarréia). Foram examinadas 276 amostras de fezes provenientes de 147 leitegadas com diarréia e 129 leitegadas sem diarréia, com idade variando entre 1 e 7 dias de vida em 28 unidades produtoras de leitões localizados no Estado do Rio Grande do Sul. Entre as amostras coletadas, 29,34% (81/276) foram positivas para pelo menos um agente pesquisado. Os coccídeos (20/276) e o C. perfringens tipo A (19/276) foram os agentes mais freqüentes. Nenhum dos enteropatógenos pesquisados obteve diferença significativa (p>0,05) entre as leitegadas caso e controle. Apenas o rotavírus (p=0,20) e o C. perfringens tipo A (p=0,16) apresentaram tendência de serem mais freqüentes em leitegadas com diarréia. Uma forte associação foi observada entre a ocorrência das diarréias e leitegadas mais novas (p<0,014). Foi observada pela primeira vez no Brasil a infecção pelo C. difficile, em 13,6% (17/132) das amostras, todavia a presença das toxinas nas fezes não teve relação com a diarréia. Os resultados obtidos indicam a necessidade de cuidados especiais quando da coleta de amostras para diagnósticos de monitoria de diarréias no período neonatal, pois é alta a chance de se obterem resultados falso positivos naqueles casos em que as doenças estiverem ocorrendo numa forma endêmica. / Diarrhea is the main clinical event occurring in pigs in the neonatal period,(as consequence of enteric disease). Besides contributing to losses in daily weight gain and feed conversion, diarrhea causes increased mortality and medication costs. The present work aimed to determine the frequency of the main viral agents (rotavirus), bacterial (E. coli, Clostridium perfringens type A and C and Clostridium difficile) and parasitic (coccidian and Cryptosporidium spp.) involved in neonatal diarrhea in case groups (with diarrhea) and controls (without diarrhea). We examined 276 fecal samples originating from 147 litters with diarrhea and 129 litters without diarrhea, with ages varying between 1 and 7 days, in 28 pig units of the state of Rio Grande do Sul, Brazil. Among the examined samples, 29.34% (81/276) were positive for at least one agent. Coccidia (20/276) and C. perfringens type A (19/276) were the most frequently isolated agents. None of the enteropathogens studied showed significant difference (p>0.05) between case and control litters. Only rotavirus (p=0.20) and C. perfringens A (p=0.16) had a tendency to present higher frequency in piglets with diarrhea. A strong association was observed between occurrence of diarrhea and litters with smaller age (p<0,014). Infection with C. difficile was diagnosed for the first time in Brazil, in 13.6% (17/132) samples; however the presence of the toxin in feces was not related to diarrhea. The present results suggest the need for special attention when sampling for the diagnosis or monitoring diarrhea in the neonatal period, as chances for obtaining false-positive results are high, especially when diseases are occurring endemically.