Orientador: Iveraldo dos Santos Dutra / Banca: Julio Cesar de Freitas / Banca: Tereza Cristina Cardoso da Silva / Resumo: A diarréia neonatal é uma das principais causas de perdas na bovinocultura. O Clostridium perfringens é um enteropatógeno amplamente distribuído na natureza e responsável por várias doenças nos animais, dentre elas a diarréia neonatal. Foram examinadas 141 amostras fecais de bezerros com diarréia e 129 amostras de animais sadios, com até 28 dias de idade e pertencentes a três rebanhos distintos. Do cultivo bacteriológico em anaerobiose foi possível isolar 36,2% e 30,2% amostras suspeitas de Clostridium perfringens dos animais enfermos e dos animais sadios, respectivamente. A genotipagem bacteriana foi efetuada empregando-se a técnica de PCR multiplex com os primers dos genes codificadores das toxinas alfa (cpa), beta (cpb), épsilon (etx), iota (itxA), enterotoxina (cpe) e toxina beta2 (cpb2). Dentre as amostras isoladas, 17/51 (33,3%) e 17/39 (43,6%) dos animais com diarréia e sadios, respectivamente, amplificaram um ou mais genes codificadores das toxinas de C. perfringens. Dos bezerros com diarréia, quatorze apresentaram somente o gene cpa (tipo A), um apresentou o cpa e cpb2 (tipo A beta2 positivo), um amplificou o cpa, itxA, e cpb2 (tipo E, beta2 positivo) e um amplificou o cpa, etx, itxA e cpb2 (tipo D e E, um ou ambos cpb2 positivo). Dentre os bezerros sadios, 10 eram exclusivamente tipo A, um era tipo A cpb2 positivo, dois eram tipo E, três eram tipo E cpb2 positivo e um era tipo D e E cpb2 positivo. Não houve correlação entre a genotipagem dos genes codificadores das toxinas de Clostridium perfringens e a presença de diarréia neonatal nos bezerros. / Abstract: Neonatal diarrhea is one of the main causes of losses in cattle herds. Clostridium perfringens is a widespread enteropathogen, and is responsible for many animal diseases such as bovine neonatal diarrhea. Fecal samples from 141 diarrheic calves and 129 healthy calves, aged up to 28 days and belonging to three herds were examined. Rates of culture positivity were 36.2% and 30.2% for diarrheic and nondiarrheic calves, respectively. Multiple isolates from primary isolation plates were subjected to simultaneous genotyping by multiplex PCR, with primers amplifying fragments of alpha (cpa), beta (cpb), epsilon (etx), iota (itxA), enterotoxin (cpe) and beta2 (cpb2) toxinencoding genes. Only 17/51 (33.3%) and 17/39 (43.6%) of these mixtures from diarrheic and nondiarrheic calves, respectively, yielded genotype information, suggesting that this may not be a viable approach to genotyping of isolates. Fourteen isolate mixtures from animals with diarrhea had only cpa (type A), one had cpa and cpb2 (type A beta2 positive), one with cpa, itxA, and cpb2 (type E, beta2 positive), and one with cpa, etx, itxA, and cpb2 (Types D and E, one or both cpb2 positive). Among 17 isolate mixtures from healthy calves, 10 were exclusively type A, one was type A cpb2 positive, two were type E, three were type E cpb2 positive, and one was types D and E cpb2 positive. There was no correlation between isolation of a given toxin type and the presence of diarrhea. / Mestre

Made available in DSpace on 2014-06-11T19:27:18Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-07-30Bitstream added on 2014-06-13T20:16:27Z : No. of bitstreams: 1 ferrarezi_mc_me_araca.pdf: 120865 bytes, checksum: 0f7d463195e9facb001a073816cb3dfd (MD5) / Universidade Estadual Paulista (UNESP) / A diarréia neonatal é uma das principais causas de perdas na bovinocultura. O Clostridium perfringens é um enteropatógeno amplamente distribuído na natureza e responsável por várias doenças nos animais, dentre elas a diarréia neonatal. Foram examinadas 141 amostras fecais de bezerros com diarréia e 129 amostras de animais sadios, com até 28 dias de idade e pertencentes a três rebanhos distintos. Do cultivo bacteriológico em anaerobiose foi possível isolar 36,2% e 30,2% amostras suspeitas de Clostridium perfringens dos animais enfermos e dos animais sadios, respectivamente. A genotipagem bacteriana foi efetuada empregando-se a técnica de PCR multiplex com os primers dos genes codificadores das toxinas alfa (cpa), beta (cpb), épsilon (etx), iota (itxA), enterotoxina (cpe) e toxina beta2 (cpb2). Dentre as amostras isoladas, 17/51 (33,3%) e 17/39 (43,6%) dos animais com diarréia e sadios, respectivamente, amplificaram um ou mais genes codificadores das toxinas de C. perfringens. Dos bezerros com diarréia, quatorze apresentaram somente o gene cpa (tipo A), um apresentou o cpa e cpb2 (tipo A beta2 positivo), um amplificou o cpa, itxA, e cpb2 (tipo E, beta2 positivo) e um amplificou o cpa, etx, itxA e cpb2 (tipo D e E, um ou ambos cpb2 positivo). Dentre os bezerros sadios, 10 eram exclusivamente tipo A, um era tipo A cpb2 positivo, dois eram tipo E, três eram tipo E cpb2 positivo e um era tipo D e E cpb2 positivo. Não houve correlação entre a genotipagem dos genes codificadores das toxinas de Clostridium perfringens e a presença de diarréia neonatal nos bezerros. / Neonatal diarrhea is one of the main causes of losses in cattle herds. Clostridium perfringens is a widespread enteropathogen, and is responsible for many animal diseases such as bovine neonatal diarrhea. Fecal samples from 141 diarrheic calves and 129 healthy calves, aged up to 28 days and belonging to three herds were examined. Rates of culture positivity were 36.2% and 30.2% for diarrheic and nondiarrheic calves, respectively. Multiple isolates from primary isolation plates were subjected to simultaneous genotyping by multiplex PCR, with primers amplifying fragments of alpha (cpa), beta (cpb), epsilon (etx), iota (itxA), enterotoxin (cpe) and beta2 (cpb2) toxinencoding genes. Only 17/51 (33.3%) and 17/39 (43.6%) of these mixtures from diarrheic and nondiarrheic calves, respectively, yielded genotype information, suggesting that this may not be a viable approach to genotyping of isolates. Fourteen isolate mixtures from animals with diarrhea had only cpa (type A), one had cpa and cpb2 (type A beta2 positive), one with cpa, itxA, and cpb2 (type E, beta2 positive), and one with cpa, etx, itxA, and cpb2 (Types D and E, one or both cpb2 positive). Among 17 isolate mixtures from healthy calves, 10 were exclusively type A, one was type A cpb2 positive, two were type E, three were type E cpb2 positive, and one was types D and E cpb2 positive. There was no correlation between isolation of a given toxin type and the presence of diarrhea.

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.

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.

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.

The effect of orally administered duodenal contents on preventing diarrhea in neonatal calves challenged with enteropathogenic E. coli was studied in a 3 x 2 factorial experiment with five replications. Newborn calves received either 0 or 200 ml of intestinal fluid inoculum, obtained from older milk-fed calves, 2 h after entering the isolation facility. Colostrum was consumed following inoculum administration. The uninoculated calves received colostrum 2 h after entering the isolation facility. In compliance with the 3 x 2 factorial arrangement, two-thirds of the calves received an E. coli challenge 12 or 24 h after colostrum feeding. The remaining calves were unchallenged. Raw milk was fed at the rate of 10 percent of body weight per day. All experimental calves were observed daily for physical condition, percent dry matter of feces, urine output, rectal temperature, and dietary intake. Body weight and packed cell volume (PCV) were determined every third day. Gamma globulin per 100 ml serum was determined at 24 h of age. The inoculum was assayed microbiologically for total anaerobes, anaerobic lactobacilli, total aerobes, coliforms, and aerobic lactobacilli. Twelve calves were slaughtered at seven days of age to determine microbiological populations of the duodenal tissue and digesta. During the first six days of life calves receiving the inoculum exhibited a lower incidence of diarrhea, greater daily urine output, lower PCV, and superior average daily gain as compared to the uninoculated calves. The incidence of diarrhea and its accompanying symptoms were most severe in uninoculated calves challenged at 12 h. Rectal temperature was not affected by treatment. The differences in response to the challenge between inoculated and uninoculated calves for the complete experimental period were similar, but not as great as during the first six days of life. Serum gamma globulin at 24 h of age was abnormally low in inoculated calves. Uninoculated calves possessed normal levels of serum gamma globulin. Bacterial populations of duodenal tissue and fluid of seven day old calves were not influenced by treatment. / M.S.

Le Canada est l’un des plus grands pays exportateurs de porc du monde et le Québec à lui seul compte pour 6% de ce commerce mondial. Pour conserver sa compétitivité et l’excellence de ses produits, une connaissance approfondie des agents infectieux circulant au sein des troupeaux est primordiale. Plusieurs pathogènes sont peu étudiés et pourtant retrouvés chez les porcs à travers la planète. Cette étude avait pour but l’évaluation de la prévalence des astrovirus porcins, calicivirus, kobuvirus porcin, rotavirus, torque teno sus virus ainsi que le virus de l’hépatite E lors du suivi de porcelets dans un réseau de production porcine, de la maternité jusqu’en fin d’engraissement. Nous voulions brosser un portrait de l’excrétion de ces virus à travers les différentes étapes de production des animaux. L’échantillonnage de porcelets sains et en diarrhée en pré-sevrage a permis de déterminer lesquelles de ces infections virales constituaient des facteurs de risque pour la diarrhée à ce stade de production. Le virome intestinal, la partie virale du microbiome, a également été caractérisé permettant de connaître la diversité des virus entériques porcins aux différentes étapes de production, ainsi qu’entre les porcelets sains et en diarrhée. De plus, la dissémination du virus de l’hépatite E dans l’environnement ainsi que les sources probables de contamination ont été décrites à l’aide d’échantillons provenant des environnements intérieurs et extérieurs de fermes d’engraissement, de la cour d’un abattoir et de transporteurs d’animaux. Les résultats obtenus ont permis de décrire les dynamiques temporelles d’excrétion de ces virus entériques porcins en fonction des stades de production des porcs, démontrant une différence dans l’excrétion de ces virus en fonction de l’âge. Les calicivirus, ainsi que les astrovirus porcins groupes 3 et 5 étaient des facteurs de risque de diarrhée en maternité. Pour la première fois au Canada, la détection et la caractérisation des souches du kobuvirus porcin ont été réalisées, permettant de mieux comprendre leur diversité et leur persistance à travers les stades de production. La diversité du virome entérique porcin a été analysée avec la plateforme de séquençage MiSeq et cette diversité était différente entre les porcelets sains et en diarrhée, ainsi qu’entre les stades de production. Cependant, les traitements enzymatiques utilisés pour le prétraitement des échantillons fécaux ne permettaient pas le séquençage de certains virus à ARN simple-brin. Des souches similaires du virus de l’hépatite E étaient présentes dans l’environnement des fermes, ainsi qu’aux endroits communs à forte circulation des intervenants du réseau. Les activités dans la cour de l’abattoir pourraient donc être impliquées dans la dissémination de ce virus. Cette étude a permis de mieux connaitre la prévalence et la distribution des virus entériques infectant les porcs. De plus, certains des virus entériques étudiés ont été reconnus comme facteurs de risque de la diarrhée en co-infections et devront être étudiés en détail pour comprendre leurs mécanismes en relation avec la diarrhée néonatale. Des interventions plus spécifiques lors d’éclosion de diarrhées porcines, dont l’étiologie est inconnue, pourront donc être réalisées, ainsi que l’élaboration de mesures de biosécurité plus adaptées en fonction du stade de production des porcs. / Canada is a major pork exporter around the world and the province of Quebec alone accounts for 6% of this trade. To maintain the province’s competitiveness and the excellence of its products, a comprehensive understanding of the infectious agents circulating in herds is essential. Several pathogens have been intensively studied, while others have yet to be investigated even though they have been reported in pigs all around the world. This study evaluated the prevalence of porcine astroviruses, calicivirus, porcine kobuvirus, rotavirus, torque teno sus virus and hepatitis E virus, monitored in a pig production network, from the nursing farms to the end of the fattening farms to portray the excretion patterns of these viruses through the different life stages of pigs. The sampling of healthy piglets alongside piglets with diarrhea in the nursing farms allowed to determine which viruses, or co-infections of viruses were factors of diarrhea at this life stage. The intestinal virome, the viral part of the microbiome, was characterized and viral diversity of porcine enteric viruses at different life stages, as well as between healthy and diarrheic piglets were evaluated. Moreover, the dissemination of the hepatitis E virus in the farm environment, as well as the possible sources of contamination were described, from the indoor and outdoor environment of fattening farms, the slaughterhouse yard and animal transporters. The results obtained in this study described the temporal excretion dynamics of these porcine enteric viruses according to the life stages of the pigs, demonstrating the difference in the excretion of the studied viruses according to the life stage. The calicivirus, as well as the porcine astrovirus groups 3 and 5 were found to be risk factors for diarrhea in the nursing farms. For the first time in Canada, the detection and characterization of porcine kobuvirus strains were evaluated and provided a better understanding of their diversity and the persistence of these strains in the network. The porcine enteric virome diversity was analyzed on a MiSeq sequencing platform and this diversity was different between healthy and diarrheic piglets, as well as between the different life stages. However, the different enzymatic treatments used as pretreatments for fecal samples altered the ability to detect certain single-stranded RNA viruses. Similar strains of the hepatitis E virus were present in the indoor and outdoor environment of the fattening farms, as well as in common places of high circulation from the various stakeholders in the pig production network. The activities in the slaughterhouse yard could therefore be involved in the spread of this virus. This study shed light on enteric viruses infecting pigs. In addition, some of the infections from enteric viruses studied were risk factors for diarrhea in co-infections and will need to be studied in more details to understand their mechanisms, in relation to neonatal diarrhea. More specific interventions during outbreaks of porcine diarrhea of unknown etiology could be carried out, as well as the development of more adapted biosecurity measures according to the life stage of the pigs.