Made available in DSpace on 2014-07-29T15:07:52Z (GMT). No. of bitstreams: 1 Dissertacao2008_Eliete_Souza.pdf: 988968 bytes, checksum: 83e526f4061a2613940db4e37b3c6955 (MD5) Previous issue date: 2009-07-25 / This study was conducted at the Experimental Center of Poultry Diseases and Laboratory of Bacteriology Department of Veterinary Medicine Veterinary School, Federal University of Goias to evaluate the effects of lactulose in Salmonella typhimurium in broilers. The experimental design was completely randomized, evaluating substance applied (lactulose) and bacterial challenge. We used 630 chicks of commercial strain, which were distributed in six treatments with seven replicates and 15 birds per experimental unit. The microbial challenge was provided to birds in the first days of life containing 5.0 X 102 CFU / 0.5 mL of Salmonella typhimurium. And lactulose at a dose of 0.023 mL / g of body weight up to 14 days of age. The weights of the birds and feed intake were seen weekly for performance measurement. On days seven, 14, 21 and 28, one bird per treatment was necropsied, and liver collected for biometric analysis and histopathological. The contents of ingluvie and caeca were submitted to enumeration of Salmonella and Escherichia coli and bacterial analysis, as well as the spleen, liver and heart were examined bacteriologically. Was also measured the pH of ingluvie, small intestine and cecum and blood collected for evaluation of liver enzymes. The quantitative performance data, biometrics liver, pH, bacterial count and liver enzymes were analyzed by applying ANOVA and Tukey (5%), and data resulting from analysis of fecal excretion of Salmonella were analyzed using the nonparametric chi-square . There was greater weight gain and lower feed conversion (P <0.05) in groups receiving lactulose with or without S. Typhimurium to 21 days of life and decreased fecal excretion of S. Typhimurium at 10 days of age for birds fed lactulose (P <0.05) from the first days of life. It was also found that the lactulose caused decrease in values (P <0.05) pH in the organs of the digestive system for seven days of life and that lactulose decreased (P <0.05) the CFU of E. coli in ingluvie at 21 and 28 days and S. Typhimurium in the treatments administered lactulose before the pathogen and ages. Lactulose prevented infection in the early period of life for birds, showing a better effect when administered 48 hours before inoculation. It was also that there was an increase in relative liver weight by S. Typhimurium in all ages studied and that the lactulose caused mild liver abnormalities, and it was also found that the S. Typhimurium did not cause severe clinical signs or mortality and that its recovery rate decreased with age. It can be concluded that lactulose causes greater weight gain in birds until one week after delivery, as well, was able to decrease the fecal excretion of S. Typhimurium. It also appears that the lactulose changed the pH values, except pH and reduced cecal colonization of S. Typhimurium in the cecum and the number of CFU of E. coli in ingluvie. It is also concluded that lactulose was able to prevent systemic infection while being provided, showing better preventive effect, and also ordered discrete liver, a process similar to congestive / O presente trabalho foi conduzido no Núcleo Experimental de Doenças de Aves e Laboratório de Bacteriologia do Departamento de Medicina Veterinária da Escola de Veterinária da Universidade Federal de Goiás para avaliar os efeitos da lactulose na Salmonella Typhimurium em frangos de corte. O delineamento utilizado foi inteiramente casualizado, avaliando substância aplicada (lactulose) e desafio bacteriano. Foram utilizados 630 pintos de linhagem comercial, os quais foram distribuídos em seis tratamentos, com sete repetições e 15 aves por unidade experimental. O desafio microbiano foi fornecido às aves no primeiro dia de vida contendo 5,0 X 102 UFC /0,5mL de Salmonella Typhimurium. E lactulose na dose de 0,023 mL/g de peso vivo até 14 dias de idade. Os pesos das aves e ração consumida foram considerados semanalmente para cálculo do desempenho. Nos dias sete, 14, 21 e 28, uma ave por parcela foi necropsiada, sendo coletado o fígado para análises biométricas e histopatológicas. Os conteúdos dos inglúvios e dos cecos foram submetidos à enumeração de Salmonella e Escherichia coli e às análises bacterianas, assim como o baço, o fígado e o coração foram examinados bacteriologicamente. Também foi aferido o pH do inglúvio, intestino delgado e ceco e coletado sangue para avaliação das enzimas hepáticas. Os dados quantitativos de desempenho, biometria do fígado, pH, contagem de bactérias e enzimas hepáticas foram analisados aplicando ANOVA e Tukey (5%), e os dados resultantes das análises de excreção fecal da Salmonella foram analisados utilizando o teste não paramétrico qui-quadrado. Observou-se maior ganho de peso e menor conversão alimentar (P<0,05) nos grupos que receberam a lactulose com ou sem S. Typhimurium até 21 dias de vida e que houve redução da excreção fecal de S. Typhimurium aos 10 dias de idade, para as aves que receberam a lactulose (P<0,05) desde o primeiro dia de vida. Constatou-se também, que a lactulose determinou redução nos valores (P<0,05) de pH nos órgãos do sistema digestivo aos sete dias de vida e que a lactulose reduziu (P<0,05) as UFC de E. coli no inglúvio aos 21 e 28 dias de vida e de S. Typhimurium nos tratamentos em que se administrou a lactulose antes do patógeno e nas idades avaliadas. A lactulose impediu infecção no período inicial de vida das aves, mostrando melhor efeito, quando administrada 48 horas antes da inoculação do patógeno. Constatou-se, ainda, que houve aumento do peso relativo do fígado pela S. Typhimurium em todas as idades estudadas e que a lactulose causou discretas alterações hepáticas; e constatou-se também, que a S. Typhimurium não determinou sinais clínicos severos, nem mortalidade e que seu índice de recuperação diminuiu com a idade. Pode-se concluir que a lactulose propiciou maior ganho de peso nas aves até uma semana após seu fornecimento; assim como, foi hábil em diminuir a excreção fecal de S. Typhimurium. Conclui-se também, que a lactulose alterou os valores de pH, com exceção do pH cecal e reduziu a colonização de S. Typhimurium no ceco e o número das UFCs de E. coli no inglúvio. Conclui-se ainda, que a lactulose foi capaz de impedir a infecção sistêmica enquanto estava sendo fornecida, tendo mostrado melhor efeito preventivo; e ainda, determinou alterações hepáticas discretas, semelhantes a um processo congestivo

Made available in DSpace on 2014-07-29T15:07:53Z (GMT). No. of bitstreams: 1 Dissertacao Hilari _Pdf.pdf: 539492 bytes, checksum: e0488461a67ab589969fbb878d006e8c (MD5) Previous issue date: 2010-02-26 / The wild animal trafficking is the third largest illegal activity in the world, ranking only behind the trafficking of weapons and drugs. The birds are the animals most affected by the illegal trade. Besides damaging to biodiversity, trafficking can also pose risks to human health. A number of diseases can be transmitted and acquired by the birds, the most commonly detected in the bacterial etiology. Improper management, especially related to transportation and overcrowding, promotes increased susceptibility to infections of birds or even the activation of latent infection with subsequent spread of pathogens. For these reasons this study aimed to conduct research and determine the Enterobacteriaceae resistance of strains of Escherichia coli, as well as raising the frequency of Chlamydophila spp. of parrots seized in actions to combat trafficking of wild animals in Goiás, with proper authorization from the Brazilian Institute of Environment and Renewable Natural Resources (IBAMA / GO). For this, 300 parrots in a period of one year were enrolled in the Screening Center of Wild Animals (CETAS), in Goiânia, Goiás, and underwent clinical and laboratory examinations. For Research Enterobacteriaceae were collected excreta from the bottom of cages, lined with aluminum foil and packed in ice for transport to the laboratory where they were examined by conventional bacteriological method. For the presence of Chlamydophila psittaci 300 cloacal swabs and 300 tracheids were collected and analyzed by polymerase chain reaction (PCR). We have identified the following excreta bacteria of the Enterobacteriaceae: Escherichia coli, 172/300 (33.87%), Enterobacter spp. 153/300 (30.12%), Klebsiella spp. 89/300 (17, 73%), Citrobacter spp. 9 / 300 (11.71%), Proteus vulgaris 21/300 (4.23%), Providencia alcalifaciens 5 / 300 (0.98%), Serratia sp.5/300 (0.98%), Hafnia aivei 3 / 300 ( 0.59%) and Salmonella sp. 1 / 300 (0.19%). Escherichia coli isolates were tested using the sensitivity to antibiotics, which revealed: amoxicillin (10mg) (70.93%), ampicillin (10mg) (75.58%), ciprofloxacin (5&#956;g) (69.76%), chloramphenicol (30&#956;g) (33.14% ), doxycycline (30&#956;g) (64.53%), enrofloxacin (5&#956;g) (41.28%), tetracycline (30&#956;g) (69.19%), sulfonamide (300&#956;g) (71.51%) of antimicrobial resistance. Of samples, 11/300 (3.66%) were positive in PCR analysis for Chlamydophilla spp. The results suggest that parrots from illegal trade are potential backers of zoonotic agents, and also point to the possibility that wild birds would constitute support for transfer of resistant phenotypes of E. coli to the human microbiota and other animals / O tráfico de animais silvestres é a terceira maior atividade ilegal do mundo, estando apenas atrás do tráfico de armas e de drogas. As aves são os animais mais atingidos pelo comércio ilegal. Além de prejudicial à biodiversidade, o tráfico também pode implicar riscos à saúde humana. Uma série de doenças podem ser transmitidas e adquiridas pelas aves, sendo as mais comumente detectadas as de etiologia bacteriana. O manejo inadequado, principalmente relacionado ao transporte e superpopulação, favorece o aumento da susceptibilidade das aves às infecções ou mesmo a ativação de infecções latentes com conseqüente disseminação de patógenos. Pelo exposto objetivouse realizar a pesquisa de Enterobacteriaceae e determinar o perfil de resistência das cepas isoladas de Escherichia coli, assim como, levantamento da freqüência de Chlamydophila spp. de psitacídeos apreendidos nas ações de combate ao tráfico de animais selvagens em Goiás, com devida autorização do Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis (IBAMA/GO). Para tanto, 300 psitacídeos, num período de um ano, foram cadastrados no Centro de Triagem de Animais Silvestres (CETAS), na cidade de Goiânia-Goiás, e submetidos a exames clínicos e laboratoriais. Para a pesquisa de Enterobacteriaceae foram colhidos excretas do fundo de gaiolas, forradas com papel alumínio e acondicionados em gelo para transporte ao laboratório, onde foram analisadas pelo método bacteriológico convencional. Para a pesquisa da Chlamydophila psittaci 300 suabes cloacais e 300 traqueais foram coletados e analisados pela reação em cadeia da polimerase (PCR). Identificaram-se nas excretas as seguintes bactérias da família Enterobacteriaceae: Escherichia coli, 172/300 (33,87%), Enterobacter spp. 153/300 (30,12%), Klebsiella spp. 89/300 (17, 73%), Citrobacter spp. 9/300 (11,71%), Proteus vulgaris 21/300 (4,23%), Providencia alcalifaciens 5/300 (0,98%), Serratia sp.5/300 (0,98%), Hafnia aivei 3/300 (0,59%) e Salmonella sp. 1/300 (0,19%). Isolados de Escherichia coli foram submetidos ao teste de sensibilidade à antimicrobianos, onde se obteve: amoxicilina (10&#956;g) (70,93%), ampicilina (10&#956;g) (75,58%), ciprofloxacina (5&#956;g) (69,76%), cloranfenicol (30&#956;g) (33,14%), doxiciclina (30&#956;g) (64,53%), enrofloxacina(5&#956;g) (41,28%), tetraciclina (30&#956;g) (69,19%), sulfonamida (300&#956;g) (71,51%) de resistência aos antimicrobianos testados. Das amostras colhidas, 11/300 (3,66%) foram positivas na análise pela PCR para Chlamydophilla spp. Os resultados sugerem que psitacídeos provenientes do comercio ilegal são potenciais veiculadores de agentes zoonóticos, e apontam ainda a possibilidade de aves selvagens se constituírem em suporte de transferência de fenótipos de E.coli resistentes para a microbiota humana e de outros animais

Chapter Introduction Salmonellae are gram negative bacteria that cause gastroenteritis and entericfever. S. enterica is divided into seven phylogenetic groups, subspecies 1, 2,3a, 3b, and 4, 6, 7. Subspecies1 includes 1,367 serovars, some of which are commonly isolated from infected birds and mammals. The other subspecies mainly colonize cold blooded animals. Salmonella typhimurium, Salmonella typhiandSalmonella enteritidis are some of the serovars, which belong to s.enterica species. S. typhimurium is one of the important causes for food poisoning in humans. It causes typhoid like fever in mice. In immuno compromised patients the infection is often fatal if it is not treated with antibiotics. Clinical features of food poisoning include abdominal pain, vomiting, nausea, abdominal cramps, dehydration etc. S. typhi causes typhoid fever in humans. No other host has been identified for this serovar. Main source of infection is contaminated food and water. No age is exempted but it is less common before2 years. Incubation period is 360 days. Clinical features include stepladder type fever, malaise, headache, hepato splenomegaly, coated tongue, Neutrogena etc. It may be fatal if untreated. Among the serovars of Salmonella infecting humans S. typhimurium and S. typhi are the most important. While S. typhimurium infects many host species including birds and mammals, S. typhi is single host adapted and infects only human. The single host adaptation of S. typhi presents it with the need for establishing are servoir of infection in the community which can serve as a source of fresh infection. Also the single host adaptation of S. typhi has made it a highly specialized pathogen which has evolved certain unique genes needed for human colonization at the same time has lost a set of genes which are needed for survival in other hosts and in the highly variable external environment. This has led to the accumulation of a vast number of pseudo genesin S. Typhi. A comparative study of the two serovars is useful in many ways. Due to varied host defense systems encountered by the two serovars owing to different niche of infection the bacterial counter defense mechanisms are also different. By focusing on the differences between genes involved in the bacterial defense of host immune response we can decipher the role played by various genes in combating the antibacterial host response. Chapter 2 The role of TolA and peptidoglycan modification in detergent resistance of pathogenic Salmonella The major Salmonella serovars that infect human are Salmonella enterica serovar Typhi (S.typhi) which cause systemic typhoid and Salmonella enterica serovar Typhimurium (S. typhimurium) which cause gastro enteritis. S. typhi resides in the gall bladder during chronic infection and S .typhimurium infects intestine .Thus both pathogens encounter high concentrations of bile and have developed mechanisms to counter it. The Tol Pal complex spanning the outermembrane and the inner cytoplasmic membrane plays an important role in maintaining the stability of the outer membrane and providing detergent resistance. The tolA gene of S. Typhi Is shorter by 27 aminoacid than S. typhimurium. The tolA gene knockout of S. typhimurium and S. typhi differed in their tritonX resistance behavoiur, morphology and low osmolality tolerance. S. typhi tolA was unable to complement the tolA defect in S. typhimurium which could probably due to the difference in the peptidoglycan layer. An analys is of the peptidoglycan modifying genes of both the serovars revealed that dacD, pbgP, ynhG are different. dacD, pbgP genes are pseudogenes in S. typhi and ynhG has a major deletion in S. typhi. Further studies reveal that a double knockout of dacD and pbpG in S. typhimurium makes it sensitive to low osmolality similar to S. typhi. Based on these results we propose a mechanism, where shortening of TolA increases detergent resistance by bringing the outer membrane into closer contact with the peptidoglycan layer, but this is achieved at the cost of reduced Lpp (Bruan’slipoprotein) peptidoglycan linkage which plays a major role in low osmolality tolerance. The pathogen S. typhi is highly adapted to the human host and cannot infect any other host. The single host adaptation and the need to survive in high concentrations of bile have made S. typhi to acquire higher bile resistance at the cost of lowered osmotic tolerance through shortening TolA and reduced Lpp and peptidoglycan binding. Chapter 3 Development of a DNA vaccine against Salmonella The immune response against Salmonella is multifaceted involving both the innate and the adaptive immune system. The characterization of specific Salmonella antigens inducing immune response could critically contribute to the development of epitope based vaccines for Salmonella. We have tried to identify aprotective Tcellepitope (s) of Salmonella, as cell mediated immunity conferred by CD8+T cells is the most crucial subset conferring protective immunity against Salmonella. It being a proven fact that secreted proteins are better in inducing cell mediated immunity than cell surface and cytosolic antigens, we have analyzed all the GenBank annotated Salmonella pathogenicity island 1 and 2 secreted proteins of S. typhimurium and S. typhi. They were subjected to BIMAS and SYFPEITHI analysis to map MHCI and MHC II binding epitopes. The huge profile of possible T cell epitopes obtained from the two classes of secreted proteins were tabulated and using a scoring system that considers the binding affinity and promiscuity of binding to more than one allele, SopB and SifB were chosen for experimental confirmation in murine immunization model. The entire Sop Band SifB genes were cloned into DNA vaccine vectors and were administered along with live attenuated Salmonella and it was found that SopB vaccination reduced the bacterial burden of organs by about 5fold on day4 and day8 after challenge with virulent Salmonella and proved to be a more efficient vaccination strategy than live attenuated bacteria alone. Chapter 4 PCR based diagnosis and Serovar Determination of Blood Borne Salmonella Typhoid fever is becoming an ever increasing threat in the developing countries. We have improved considerably upon the existing PCR based diagnosis method by designing primers against a region which is unique to S. typhiand S. paratyphiA, corresponding to the gene STY0312 in S. typhi and its homolog SPA2476 in S. paratyphiA. An additional set of primers amplify another region in S. typhi CT18 and S. typhiTy2 corresponding to the region between the genes STY0313 toSTY0316 but which is absent in S.paratyphi A. The threat of false negative result arising due to mutation in hypervariable genes has been reduced by targeting a gene unique to typhoidal Salmonella as a diagnostic marker. The amplified region has been tested for genomic stability by amplifying them from clinical is olates of patients from various geographical locations in India, there by showing that this region is potentially stable. These set of primers can also differentiate between S. typhiCT18, S. typhiTy2 and S. paratyphi A which have stable deletions in this specific locus. The PCR assay designed in this study has a sensitivityof95%ascompared to the Widal test which had only 63%. As observed, in certain cases the PCR assay was more sensitive than the blood culture test as the PCR based detection could also detect dead bacteria.

This diploma thesis is focused on the evaluation of six selected zoonoses with the occurrence in South Bohemia (campylobacteriosis, salmonellosis, listeriosis, tick-borne meningoencephalitis, Lyme borreliosis, tularemia) between the years 2003-2013. Campylobacteriosis and salmonellosis were evaluated the most common zoonoses in the South Bohemian Region during the monitored period. Their common feature is the alimentary transmission. Between 2003 to 2008 salmonellosis recorded the highest number of reported cases, on the contrary, campylobacteriosis has reported the highest number of occurrences since 2008. District of Czech Budweis showed the highest incidence of both zoonoses during the monitored period (4,139 cases of salmonellosis and 4,924 cases of campylobacteriosis). Another but not less important zoonotic is tick-borne meningoencephalitis, which incidence had the highest number in South Bohemian Region of all the regions of Czech Republic. The second part of the thesis is based on questionnaire studies focused on awareness of zoonoses and the issue of resistance and overuse of antibiotics. In total 479 questionnaires were evaluated. The survey findings may be equally described as interesting and positive, since the respondents had considerable awareness of the issue of antibiotic resistance.