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

A series of experiments were conducted to examine the effect of chemical composition of the diet on intestinal <i>Clostridium perfringens</i> populations and necrotic enteritis (NE) in broiler chickens. In the first experiment, birds were fed high concentrations of dietary protein (fish meal or soy protein concentrate) and soluble fiber (guar gum). Clinical NE was not observed, however, there was a high level of <i>C. perfringen</i> colonization especially in guar gum fed birds. The next set of experiments examined the effect of various levels of DL-Met or MHA-FA on <i>C. perfringen</i> and other intestinal microbes. These experiments demonstrated a significant reduction (P < 0.05) in <i>C. perfringen</i> growth with methionine supplementation in ileum and cecum. The results suggest that both DL-Met and MHA-FA may reduce intestinal populations of <i>C. perfringen</i> in broiler chickens when used in high concentrations. The next three experiments were conducted to examine the effect of dietary glycine levels on gut <i>C. perfringen</i> populations, α-toxin production and NE lesion scores. Majority of birds showed clinical signs of disease with 4.16-8.33% mortality. There was a direct correlation between intestinal <i>C. perfringen</i> populations, NE lesions scores and mortality with dietary glycine level. However, due to the use of gelatin as the dietary source of glycine in these experiments, the diets also contained high proline levels which confounded our results. The last study was conducted to establish a direct causative relationship between dietary glycine concentration and <i>C. perfringen</i> growth and/or NE in broiler chickens using encapsulated amino acids. Birds fed diets containing high levels of encapsulated glycine had higher NE lesion scores than those fed encapsulated proline or no encapsulated amino acids, thus demonstrating a direct effect of glycine on intestinal <i>C. perfringen</i> growth. It is concluded that amino acid composition of dietary protein is an important determinant of intestinal microbial growth, particularly <i>C. perfringen</i>, and could affect incidence of NE in broiler chickens.

Three experiments were conducted to investigate the effects of various cereal grain types on the production performance of dairy cattle and the colonization of <i>Clostridium perfringens</i> in the intestinal tract of cattle. The first experiment investigated the effect of feeding two different cultivars of barley (cv. Harrington and Valier) and two different cultivars oat (cv. Derby and AC Assiniboia) grain on feed intake, milk yield and milk constituents. It was found that barley or oat may both be successfully incorporated into the concentrate portion of western Canadian dairy rations. NRC Dairy 2001 predictions for both oat and barley diets very closely predicted dry matter intake and milk production. AC Assiniboia oat fed cows produced the lowest percentage of fat (P<0.05) compared to the other dietary treatments. Oat fed cows produced milk with lower total solids (P<0.05) than barley fed cows. Experiment two investigated the effect of replacing barley with corn in the concentrate of a dairy ration on performance parameters as well as fecal shedding of <i>C. perfringens</i>. Corn can successfully replace a portion of the cereal grain in a typical western Canadian dairy ration without adverse effects on milk production, feed intake and feed efficiency. Corn fed cows produced significantly higher milk protein than barley fed cows (P<0.05). Dairy NRC 2001 milk production predictions were similar to observed milk yields. Mean (±SEM) level of colonization (log cfu/g) of <i>C. perfringens</i> in the feces was lower (P<0.05) in barley-fed (1.30±0.27) compared with corn-fed (2.47±0.27) cows. Experiment three was a survey to determine the level of shedding of <i>C. perfringens</i> in feces of dairy cattle at different ages and stages of lactation and in feedlot cattle through the backgrounding and finishing phases. No relationship was found between heifer age and level of shedding or between days in lactation and level of shedding. Sixteen percent of the sampled cows exhibited high levels of shedding (3.02±.46) log cfu/g feces) through all stages of lactation without exhibiting adverse effects on health. No relationship was found between age of heifer and level of <i>C. perfringens</i> shedding in the feces. Steers shed significantly higher (P<0.05) levels of <i>C. perfringens</i> during the last phase of finishing compared with other feeding phases. Further research investigating <i>C. perfringens</i> colonization in cattle is required.

A series of experiments were conducted to examine the effect of chemical composition of the diet on intestinal <i>Clostridium perfringens</i> populations and necrotic enteritis (NE) in broiler chickens. In the first experiment, birds were fed high concentrations of dietary protein (fish meal or soy protein concentrate) and soluble fiber (guar gum). Clinical NE was not observed, however, there was a high level of <i>C. perfringen</i> colonization especially in guar gum fed birds. The next set of experiments examined the effect of various levels of DL-Met or MHA-FA on <i>C. perfringen</i> and other intestinal microbes. These experiments demonstrated a significant reduction (P < 0.05) in <i>C. perfringen</i> growth with methionine supplementation in ileum and cecum. The results suggest that both DL-Met and MHA-FA may reduce intestinal populations of <i>C. perfringen</i> in broiler chickens when used in high concentrations. The next three experiments were conducted to examine the effect of dietary glycine levels on gut <i>C. perfringen</i> populations, α-toxin production and NE lesion scores. Majority of birds showed clinical signs of disease with 4.16-8.33% mortality. There was a direct correlation between intestinal <i>C. perfringen</i> populations, NE lesions scores and mortality with dietary glycine level. However, due to the use of gelatin as the dietary source of glycine in these experiments, the diets also contained high proline levels which confounded our results. The last study was conducted to establish a direct causative relationship between dietary glycine concentration and <i>C. perfringen</i> growth and/or NE in broiler chickens using encapsulated amino acids. Birds fed diets containing high levels of encapsulated glycine had higher NE lesion scores than those fed encapsulated proline or no encapsulated amino acids, thus demonstrating a direct effect of glycine on intestinal <i>C. perfringen</i> growth. It is concluded that amino acid composition of dietary protein is an important determinant of intestinal microbial growth, particularly <i>C. perfringen</i>, and could affect incidence of NE in broiler chickens.

Three experiments were conducted to investigate the effects of various cereal grain types on the production performance of dairy cattle and the colonization of <i>Clostridium perfringens</i> in the intestinal tract of cattle. The first experiment investigated the effect of feeding two different cultivars of barley (cv. Harrington and Valier) and two different cultivars oat (cv. Derby and AC Assiniboia) grain on feed intake, milk yield and milk constituents. It was found that barley or oat may both be successfully incorporated into the concentrate portion of western Canadian dairy rations. NRC Dairy 2001 predictions for both oat and barley diets very closely predicted dry matter intake and milk production. AC Assiniboia oat fed cows produced the lowest percentage of fat (P<0.05) compared to the other dietary treatments. Oat fed cows produced milk with lower total solids (P<0.05) than barley fed cows. Experiment two investigated the effect of replacing barley with corn in the concentrate of a dairy ration on performance parameters as well as fecal shedding of <i>C. perfringens</i>. Corn can successfully replace a portion of the cereal grain in a typical western Canadian dairy ration without adverse effects on milk production, feed intake and feed efficiency. Corn fed cows produced significantly higher milk protein than barley fed cows (P<0.05). Dairy NRC 2001 milk production predictions were similar to observed milk yields. Mean (±SEM) level of colonization (log cfu/g) of <i>C. perfringens</i> in the feces was lower (P<0.05) in barley-fed (1.30±0.27) compared with corn-fed (2.47±0.27) cows. Experiment three was a survey to determine the level of shedding of <i>C. perfringens</i> in feces of dairy cattle at different ages and stages of lactation and in feedlot cattle through the backgrounding and finishing phases. No relationship was found between heifer age and level of shedding or between days in lactation and level of shedding. Sixteen percent of the sampled cows exhibited high levels of shedding (3.02±.46) log cfu/g feces) through all stages of lactation without exhibiting adverse effects on health. No relationship was found between age of heifer and level of <i>C. perfringens</i> shedding in the feces. Steers shed significantly higher (P<0.05) levels of <i>C. perfringens</i> during the last phase of finishing compared with other feeding phases. Further research investigating <i>C. perfringens</i> colonization in cattle is required.

The survival and germination of Clostridium perfringens spores in different heating media and at different heating rates was studied to determine the fate of C. perfringens spores during abusive cooking and cooling of pork products. The heat resistance (HR) of C. perfringens spores from three strains that were either previously heat shocked (HS) or non-heat shocked (NHS) was determined individually and as a cocktail in phosphate buffer solution (pH 7.4) (PBS), beef gravy (BG), ground pork (GP) and cured ground pork (CGP) at 75ºC. The effect of the heating rate on HR, germination and outgrowth of C. perfringens spores in CGP was determined by increasing the temperature from 20 to 75ºC at a rate of 4, 8, and 12ºC/h prior to heating and holding at 75ºC for 48 h. Heating rates at 4ºC/h in GP and CGP were repeated with additional cooling from 54.4 to 7.2ºC within 20 h (temperature abuse). Linear survival curves were observed on NHS spores in the four heating media, whereas HS spores showed linear curves when heated in PBS and BG, and biphasic curves when heated in GP and CGP. In general, HS spores were more heat sensitive than NHS spores. NHS spores heated in GP had greater HR than spores heated in CGP, BG or PBS. There were no significant differences (P>0.05) on the HR of C. perfringens spores in CGP heated from 20 to 75ºC at 4, 8, or 12ºC/h. Heating rates of 8 and 12ºC/h showed no difference in germination and outgrowth of inoculated spores, whereas at 4ºC/h, growth of C. perfringens occurred between 44 and 56ºC. Temperature abuse during cooling of GP resulted in 2.8 log CFU/g increase of C. perfringens counts. In CGP, C. perfringens counts decreased by 1.1 log CFU/g during cooling from 54.4 to 36.3ºC and then increased by 1 log CFU/g until the product reached 7.2ºC. However, with an initial inoculum in raw CGP of 5 log CFU C. perfringens spores/g, C. perfringens counts did not exceed 3.4 log CFU/g during a 20 h abusive cooling. These results suggest there is no risk associated with C. perfringens in cured pork products under the conditions tested. Results from the present study indicate that different behavior may be expected with different meat products.

La enterotoxemia, causada por el Clostridium perfringens, es la enfermedad infecciosa más importante que afecta a las alpacas, debido a que ocasiona elevadas tasas de mortalidad neonatal de hasta 70%. Recientes estudios han sugerido la participación de la Cp-PLC (C. perfringens fosfolipasa C) como factor de virulencia responsable del cuadro enterotoxemico en alpacas y otras especies domesticas. El presente estudio evaluó las características toxigénicas de la Cp-PLC y de sobrenadantes de diferentes aislados de C. perfringens obtenidos de casos de enterotoxemia en alpacas relacionándolos con sus niveles de producción de Cp-PLC. El protocolo de purificación de Cp-PLC mostró ser exitoso, mostrando su comportamiento como una enterotoxina incapaz de generar lesiones entéricas. Asimismo, los aislados de C. perfringens analizados evidenciaron distintas características toxigénicas independientes de la presencia de Cp-PLC. Al parecer, la Cp-PLC no seria un factor esencial del C. perfringens en la producción de lesiones entéricas en casos de enterotoxemias en alpacas. Palabras Claves: Cp-PLC, Clostridium perfringens, enterotoxigenico / Enterotoxemia caused by Clostridium perfringens, causes a mortality neonatal rate up to 70%, this is why it is considered as the most important infections disease. Recent studies has suggested that Cp-PLC (Clostridium perfringens phospholipase C) is a main virulence factor responsible of the enterotoxemic lesions found in alpacas and other domestic animals. This study evaluated the toxigenic characteristics of Cp-PLC and of supercultures of C. perfringens isolates from enterotoxemia in alpacas associated with their levels of Cp-PLC production. The Cp-PLC purification protocol used was successful, showing that Cp-PLC as an enterotoxin enteric unable to cause injury. Similarly, C. perfringens isolates analyzed showed different toxigenic characteristics independently of the Cp-PLC production. Apparently, Cp-PLC does not be a essential factor from C. perfringens in the production of enteric lesions in cases of enterotoxemia in alpacas. Key Word: Cp-PLC, Clostridium perfringens, enterotoxigenic.

Thesis (M. Sc.)--University of Hong Kong, 1970. / Mimeographed.

Necrotic enteritis (NE) is an economically important, but poorly understood, disease of poultry, typically caused by Clostridium perfringens Type A strains that carry the NetB toxin gene. The objective of the current research was to identify additional genes associated with NE-causing C. perfringens strains, and thus putatively involved in virulence. To identify novel NE-associated genes, the draft genome sequences of seven C. perfringens NE isolates and one isolate from a healthy chicken were compared against nine non-poultry genomes, and three highly-conserved NE-associated loci (NELoc-1 – 3) were identified. The largest locus (NELoc-1) encoded 37 putative proteins, including NetB, an internalin-like protein, a ricin-domain protein, two leukocidins, several cell-surface proteins and a cyclic-di-guanidine monophosphate (c-di-GMP) signaling system. NELoc-1 and -3 were both localized to separate plasmids that are both predicted to undergo conjugative transfer. These findings suggest that NE pathogenesis involves multiple virulence factors that are encoded on discrete pathogenicity loci, some of which are plasmid-borne. To further elucidate the genetic basis of NE pathogenicity, a microarray was developed based on two of the sequenced NE bird isolates, and used to assess the gene content of 54 isolates from chickens with and without NE. Variable genomic regions associated with netB-positive isolates were identified, including several chromosomal fitness-related loci, such as a carbohydrate ABC transporter, ferric-iron siderophore uptake system, and adhesion locus. Additional loci were related to plasmid maintenance. This study suggests that chromosomal background confers a selective advantage to NE-causing strains, possibly through mechanisms involving iron acquisition, carbohydrate metabolism and plasmid maintenance Finally, the relationship between netB presence, NetB production and host NE status was examined to assess the hypothesis that netB-positive isolates from healthy birds frequently do not express NetB toxin. The expression of NetB toxin was determined in 57 poultry isolates, demonstrating that NetB expression is closely correlated with the presence of netB, and independent of host disease status. In conclusion, these studies have identified a number of C. perfringens genes predicted to play a role in NE pathogenesis, and suggest that NE is a complex, multifactorial disease involving both host and plasmid-encoded virulence factors.

Thesis (M.S.)--University of Wisconsin--Madison, 1965. / eContent provider-neutral record in process. Description based on print version record. Bibliography: l. 78-85.