Moniliophthora perniciosa é o agente causal da doença vassoura-de-bruxa em cacaueiro (Theobroma cacao). Os sintomas da doença compreendem perda de dominância apical, inchamento e excesso de brotações em ramos novos, reversão de meristemas florais em vegetativos, partenocarpia e lesões necróticas em frutos, sugerindo a ocorrência de alterações hormonais no hospedeiro. A disponibilidade de isolados do biótipo-S capazes de infectar o tomateiro, permitiu a utilização da cultivar miniatura \'Micro-Tom\' (MT) como um modelo para estudo da interação Moniliophthora perniciosa x Solanum lycopersicum. Além de provocar sintomas característicos no MT, a disponibilidade de mutantes e linhas transgênicas introgredidos em MT, com alterações que afetam o metabolismo e sensibilidade hormonal, permitem investigar o papel dos hormônios vegetais no desenvolvimento dos sintomas. Inicialmente, foi avaliada a agressividade de três isolados do biótipo-S no MT, sendo que um isolado de Tiradentes apresentou maior agressividade, com maior incidência dos sintomas, maior engrossamento do caule, redução na altura das plantas, aumento no número de lóculos nos frutos e redução na biomassa radicular. Mutantes com alterações na percepção para auxina (diageotropica e entire) e uma linha transgênica expressando uma citocinina oxidase de Arabidopsis (35S::AtCKX2) diferiram para o engrossamento do caule e distribuição do número de lóculos nos frutos em relação ao MT. A linha transgênica 35S::AtCKX2 diferiu significativamente de MT com menor incidência de infecção. O engrossamento do caule associa-se ao aumento na área do córtex e, principalmente do xilema e floema. A aplicação exógena de citocinina sintética benzil-adenina (BA) e da auxina sintética ácido naftaleno acético (ANA) em MT evocam sintomas similares aos de plantas infectadas com M. perniciosa. Linhas transgênicas repórter de sinalização por citocinina (ARR5::GUS) ou auxina (DR5::GUS) indicaram sinalização diferencial por citocinina a 24 h e 36 h após inoculação (HAI) e 48 HAI por auxina. A infecção por M. perniciosa aumentou os níveis de ácido jasmônico, ácido salicílico (AS) e auxina em MT entre 5 d a 30 DAI, com maior incremento aos 5 DAI, enquanto que o nível de ácido abscísico aumentou aos 20 d e 30 DAI, e AS foi o único detectado em micélio dicariótico do biótipo-S. Genes de biossíntese de citocinina (IPT), ativação (LOG), degradação (CKX) e resposta à citocinina (ARRs e CRF) e auxina (AUX/IAA, ARFs, SAUR e GH3) foram induzidos em MT inoculado de 12 h a 5 DAI, mas com maior acúmulo de transcritos aos 30 DAI. M. perniciosa induziu maior expressão desses genes citados e de biossíntese auxina, nos momentos iniciais da interação (12 h a 5 DAI) em 35S::AtCKX2 do que no MT. O efeito da infecção em aumentar o número de lóculos nos frutos parece ser independente ou downstream a mutação fasciated, Mouse ears e ovate. A mutação Lanceolate parece ter um papel na redução do efeito da infecção em aumentar o número de lóculos. Os resultados obtidos sugerem que a infecção pelo M. perniciosa em MT altere os níveis/sinalização dos hormônios vegetais, principalmente auxina e citocinina, provocando o engrossamento do caule (aumento no xilema, floema e córtex), redução no crescimento e na biomassa radicular e aumento no número de lóculos nos frutos / Moniliophthora pernicisa is the causal agent of witches\' broom disease in cocoa (Theobroma cacao). The disease symptoms comprise loss of apical dominance, thickening and proliferation of axillary shoots, shift from inflorescence into vegetative meristem, parthernocarpy and necrotic lesions on fruits, suggesting a host hormonal imbalance. The availability of an isolated of S-biotype M. perniciosa, which colonizes tomato, enabled the utilization of the miniature tomato (Solanum lycopersicum) cultivar \'Micro-Tom\' (MT) as a suitable model to study the pathosystem M. perniciosa x S. lycopersicum. In addition to the characteristic symptoms of the infection in MT, the availability of mutants and transgenic lines introgessed into MT, with changes in plant metabolism and hormonal sensitivity, enable the investigation of the role of plant hormones in the development of symptoms. Initially, we evaluated the aggressiveness of three S-biotype M. perniciosa isolates. The isolate \'Tiradentes\' showed greater aggressiveness infecting MT, with higher plant infection incidence, greater stem thickening, reduction in plant height, increase in fruit locule number and reduction in root dry weight. Mutants with altered auxin perception (diageotropica e entire) and the transgenic line expressing a cytokinin oxidase gene of arabidopsis (35S::AtCKX2) differed in stem thickening and fruit locule number distribution, as compared to MT. The transgenic line 35S::AtCKX2 differed significantly from MT, showing lower incidence of infection. The thickening of the stem may be related with an increase in area of the cortex, especially xylem and phloem. The exogenous application of synthetic cytokinin benzyl adenine (BA) and auxin naphthalene acetic acid (NAA) in MT induces similar symptoms to plants infected with M. perniciosa. Cytokinin (ARR5::GUS) and auxin (DR5::GUS) signaling reporter transgenic lines revealed differential cytokinin signaling 24 h e 36 h hours after inoculation (HAI) and differential auxin signaling in 48 HAI. Infection of MT by M. perniciosa increased the content of JA, SA and auxin during the development of symptoms from 5 d to 30 DAI, with greater increase at an early stage of symptoms development (5 days after inoculation - DAI), whereas abcisic acid content increased in 20 and 30 DAI, and only AS was detected in dicariotic mycelium of the S-biotype M. perniciosa. Cytokinin biosynthesis (IPT), activating (LOG), and breakdown (CKX) genes and response to cytokynin genes (ARRs e CRF) and auxin (AUX/IAA, ARFs, SAUR e GH3) were induced in MT infected in 12 h a 5 DAI, with greater accumulation of transcripts in 30 DAI. M. perniciosa induced higher levels of IPT, LOG, CKX, ARRs, and CRF genes and auxin biosynthesis genes at an ealry stage of infection (12 h a 5 DAI) in 35S::AtCKX2, as compared to MT. The effect of the infection on increasing fruit locule number seems to be independent or downstream fasciated, Mouse ears and ovate mutation. Lanceolate mutation seems to play a role in reducing M. perniciosa ability of increasing fruit locule number. The results suggest infection of MT by S-biotpye M. perniciosa alters levels/signaling of the hormones, especially auxin and cytokinin, inducing stem thickening (increasing xylem, phloem and cortex), reduction in plant height, root dry weight and increase in fruit locule number

Entre as 12 espécies do gênero Yersinia, Yersinia enterocolitica é a mais prevalente como causa de doença em humanos e animais. Sua patogenicidade é relacionada, entre outras características, a seis biotipos: o 1B e os biotipos 2 a 5 comprovadamente patogênicos e o biotipo 1A, considerado como não-patogênico. Entretanto, dados da literatura relatam linhagens do biotipo 1A como sendo os agentes causais de infecções em humanos e animais. O objetivo deste trabalho foi investigar o potencial patogênico e verificar a similaridade genômica de linhagens de Y. enterocolitica biotipo 1A, isoladas de material clínico e não-clínico. Foram estudadas 52 linhagens de Yersinia enterocolitica biotipo 1A isoladas de humanos (11), animais (11), alimentos (15) e ambiente (15), quanto a susceptibilidade a antimicrobianos, comportamento frente a testes fenotípicos relacionados à virulência, resistência a reativos intermediários do oxigênio, invasão a células HEp-2 e Caco-2, presença de genes de virulência por PCR e similaridade genômica por Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR) e Pulsed-field gel electrophoresis (PFGE). Tanto as linhagens clínicas como as não-clínicas apresentaram resistência à ampicilina e à cefalotina. Não foi observada diferença entre linhagens de origem clínica e não-clínica frente aos testes de fermentação da salicina, hidrólise da esculina, atividade da pirazinamidase, reativos intermediários do oxigênio e invasão a células HEp-2. Entretanto, linhagens de origem não-clínica foram mais invasivas a células Caco-2 do que as de origem clínica. Oito dos 11 genes de virulência pesquisados foram encontrados. Os genes ystB, hreP e fepD foram mais freqüentemente detectados em linhagens de origem clínica. Ao contrário, os genes myfA, fepA, fes e tccC apresentaram-se mais freqüentes nas linhagens de origem não-clínica. Entretanto, a diferença na freqüência de tais genes não foi estatisticamente significativa entre linhagens clínicas e não-clínicas. O gene inv foi detectado em todas as linhagens estudadas, entretanto, os genes ail, ystA e virF não foram detectados em nenhuma das 52 linhagens. O dendrograma de similaridade genômica consenso das técnicas de ERIC-PCR e PFGE, permitiu a visualização de dois grupos (A e B). Foi observada uma alta similaridade genômica (>63%) entre quase todas as linhagens isoladas de humanos e animais, bem como uma alta similaridade genômica para a maioria das linhagens de origem clínica e não-clínica (>58%). O índice de discriminação de ERIC-PCR foi 0,98, e o de PFGE foi 0,99. Entre as linhagens do biotipo 1A estudadas, não foi observada diferença entre o potencial patogênico de linhagens de origem clínica e não-clínica frente aos testes fenotípicos realizados e prevalência de genes de virulência pesquisados. A exceção foi o teste de invasão a células Caco-2, onde as linhagens não-clínicas foram mais invasivas. As técnicas de ERIC-PCR e PFGE discriminaram similarmente as linhagens estudadas. A alta similaridade genômica entre as linhagens de origem clínica e não-clínica evidencia os animais como sendo importantes reservatórios de Y. enterocolitica biotipo 1A e sugere que isolados de ambiente e alimentos tem sido fonte de contaminação de humanos e animais. / Among the 12 species of the genus Yersinia, Yersinia enterocolitica is the most prevalent cause of illness in humans and animals. Among other characteristics, its patogenicity is related to six biotypes: 1B and 2 to 5 considered pathogenic and the 1A biotype considered non-pathogenic. Despite being defined as non-pathogenic, literature has been shown that biotype 1A strains may be the etiological agents of infections in humans and animals. The aim of this work was to investigate the pathogenic potential and to verify the genomic similarity of Y. enterocolitica biotype 1A isolated from clinical and non-clinical sources. Fifty-two strains of Y. enterocolitica biotype 1A isolated from humans (11), animals (11), food (15), and environment (15) were analyzed regarding their susceptibility to antimicrobials, behavior against phenotypic tests related to virulence, resistance to oxygen intermediate reactives, invasion to HEp-2 and Caco-2 cells, presence of virulence genes by PCR, and genomic similarity by Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR) and Pulsed-field gel electrophoresis (PFGE). Both clinical and non-clinical strains showed resistance to ampicillin and cephalothin. It was not observed any difference in the pathogenic potential between clinical and non-clinical strains face of the following tests: salicine fermentation, esculin hydrolysis, pirazinamidase activity, oxygen intermediate reactives and HEp-2 cell invasion assay. On the other hand, the non-clinical strains were more invasive to Caco-2 cells than the clinical ones. Eight of 11 studied virulence genes were found. Genes ystB, hreP and fepD were more often detected in clinical strains. In contrast, myfA, fepA, fes and tccC were presented more frequently in non-clinical strains. However, the frequency difference of those genes was not statistically significant between clinical and non-clinical strains. The inv gene was detected in all the strains studied; but no ail, ystA, and virF genes were found in any of the 52 strains. ERIC-PCR and PFGE dendogram allowed the visualization of two groups named A and B. It was observed a high genomic similarity among almost all human and animal isolated strains (>63%), as well as a high genomic similarity between the clinical and non-clinical strains (>58%). The discriminatory index for ERIC-PCR was 0.98 and for PFGE was 0.99. Among biotype 1A strains no difference was observed between the pathogenic potential of clinical and non-clinical strains face to the phenotype tests employed, and regarding the prevalence of the studied virulence genes. The exception was the Caco-2 cells invasion assay where non-clinical strains were more invasive., ERIC-PCR and PFGE discriminated the studied strains similarly. The high genomic similarity between the clinical and non-clinical strains gives evidence that animals constitute important reservoirs of Y.enterocolitica biotype 1A and suggests that environmental and food isolates have been the source of human and animal infections.

Dentre as espécies do gênero Yersinia, Yersinia enterocolitica é a espécie mais prevalente como causa de doença em humanos e animais. Y. enterocolitica é dividida em seis biotipos. Os biotipos 1B, 2, 3, 4 e 5 compreendem linhagens associadas à doença em humanos e animais, enquanto o biotipo 1A consiste de linhagens consideradas não patogênicas. Apesar de Y. enterocolitica biotipo 2 ser de importância clínica, há uma escassez de estudos no país, o que dificulta avaliar o envolvimento dessa bactéria como causa de doença em humanos e em animais, bem como, determinar o impacto de sua presença no meio-ambiente. O objetivo deste trabalho foi investigar o potencial patogênico, determinar o perfil de suscetibilidade a antimicrobianos e verificar a diversidade genotípica de linhagens de Y. enterocolitica biotipo 2 isoladas no Brasil. Foram estudadas 40 linhagens de Y. enterocolitica biotipo 2, isoladas de humanos (5), ambiente (34) e animal (1), entre os anos de 1979 e 1998. Ademais, nas análises filogenéticas, foram acrescidas 26 linhagens de Y. enterocolitica pertencentes aos outros biotipos, com o intuito de comparar as linhagens de Y. enterocolitica biotipo 2 aos biotipos 1A, 1B, 3, 4 e 5. As linhagens de humanos e animal foram sensíveis a todos os 14 antimicrobianos testados. Dentre as 34 linhagens de ambiente, sete (20,6%) foram resistentes a um ou dois antimicrobianos, sendo esses, amicacina, cefoxitina, gentamicina, e sulfametoxazol - trimetoprima. Todas as linhagens apresentaram os genes inv, ail, ystA, hreP, tccC e myfA. Os genes fepD e fes foram detectados em 39 (97,5%) linhagens, o gene virF foi encontrado em três (7,5%) linhagens, os genes ystB e fepA não foram detectados em nenhuma linhagem. Todas as linhagens apresentaram comportamento relacionado à virulência frente aos testes fenotípicos de atividade da pirazinamidase, hidrólise da esculina e fermentação da salicina. O dendrograma de similaridade genética de Enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) agrupou as linhagens de Y. enterocolitica biotipo 2 em cinco grupos denominados A, B, C, D e E. Todas as linhagens, com exceção de duas, apresentaram similaridade genética superior a 88,3%. O dendrograma de similaridade genética de Pulsed field gel electrophoresis (PFGE) agrupou as linhagens de Y. enterocolitica biotipo 2 em três grupos denominados I, J e K. A maioria das linhagens (72,5%) apresentou similaridade ii genética superior a 78,3%. O dendrograma de similaridade genética de Multilocus variable number tandem repeat analysis (MLVA) agrupou as linhagens de Y. enterocolitica biotipo 2 em dois grupos denominados O e P com similaridade genética superior a 37,7%. Pode-se concluir que o potencial patogênico das linhagens de Y. enterocolitica biotipo 2 foi evidenciado pela prevalência da maioria dos marcadores de virulência, bem como, pelo comportamento relacionado à virulência frente aos testes fenotípicos pesquisados. Algumas linhagens apresentaram-se resistentes a antimicrobianos de primeira escolha no tratamento de yersiniose, o que pode acarretar em falha terapêutica. Os resultados de ERIC-PCR e PFGE mostraram a alta similaridade entre as linhagens de Y. enterocolitica biotipo 2, sugerindo que as mesmas pouco se diferenciaram ao longo dos 19 anos e que possivelmente o meio ambiente tem sido uma fonte de contaminação para humanos e animais no Brasil. A técnica de MLVA agrupou as linhagens de Y. enterocolitica biotipo 2 quanto à sua origem e a técnica de ERIC-PCR agrupou as linhagens de Y. enterocolitica biotipos 1A, 1B, 2, 3, 4, e 5 quanto às diferentes patogenicidades características de cada biotipo. / Among the species of the genus Yersinia, Yersinia enterocolitica is the most prevalent species that cause illness in humans and animals. Y. enterocolitica is divided into six biotypes. Biotypes 1B, 2, 3, 4 e 5 comprise strains associated to illness in humans and animals, while biotype 1A comprise strains considered nonpathogenic. Despite of the fact that Y. enterocolitica biotype 2 is of clinical importance, there is a paucity of studies in this country, which makes difficult to assess the involvement of this bacteria as a cause of illness in humans and animals, as well as to determine the impact of its presence in the environment. The aim of this work was to investigate the pathogenic potential, to determine the antimicrobial resistance profile and to verify the genetic diversity of Y. enterocolitica biotype 2 strains isolated in Brazil. Forty strains of Y. enterocolitica biotype 2 isolated from humans (5), environment (34) and animal (1), between 1979 and 1998 were studied. Besides, in the phylogenetic analyzes it was added 26 Y. enterocolitica strains belonging to the other biotypes, in order to compare the Y. enterocolitica biotype 2 strains to biotypes 1A, 1B, 3, 4 e 5. Humans and animals strains showed susceptibility to all 14 antibiotics tested. Among the 34 environment strains, seven (20.6%) were resistant to one or two antibiotics used such as amikacin, cefoxitin, gentamicin and sulfamethoxazole-trimethoprim. All the strains presented the genes inv, ail, ystA, hreP, tccC and myfA. Genes fepD and fes were detected in 39 (97.5%) strains, virF was found in three (7.5%) strains, and ystB and fepA were not detected in any strains. All the strains exhibited behavior related to virulence against the phenotypic tests of pyrazinamidase activity, esculin hydrolysis and salicin fermentation. The dendrogram of genetic similarity of Enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) grouped the Y. enterocolitica biotype 2 strains in five groups, designated A, B, C, D and E. All the strains, except two, showed a genetic similarity of more than 88.3%. The dendrogram of genetic similarity of Pulsed field gel electrophoresis (PFGE) grouped the Y. enterocolitica biotype 2 strains in three groups, designated I, J and K. The majority of the strains (72.5%) showed a genetic similarity of more than 78.3%. The dendrogram of genetic similarity of Multilocus variable number tandem repeat analysis (MLVA) grouped the Y. enterocolitica iv biotype 2 strains in two groups, designated O and P with a genetic similarity of more than 37.7%. It is possible to conclude that the pathogenic potential of the Y. enterocolitica biotype 2 strains was highlighted by the prevalence of the majority of the virulence markers searched, as well as by the behavior related to virulence against the phenotypic tests. Some strains were resistant to antimicrobials that are the first choice for yersiniosis treatment, which can result in therapeutic failure. The results of ERIC-PCR and PFGE showed a high genetic similarity between the Y. enterocolitica biotype 2 strains, suggesting that the strains differed little over 19 years, and that the environment has been possibly a source of humans and animals infections in Brazil. The MLVA technique grouped the Y. enterocolitica biotype 2 strains according their origins, and the ERIC-PCR technique grouped the Y. enterocolitica biotypes 1A, 1B, 2, 3, 4 and 5 strains according to the different pathogenicity characteristics of each biotype.

Made available in DSpace on 2017-07-21T19:59:59Z (GMT). No. of bitstreams: 1 Franciane Gomig.pdf: 1491620 bytes, checksum: 9e4413251eb0f4e70622124b18ded1a5 (MD5) Previous issue date: 2013-04-16 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Urinary tract infections show a high incidence in the population, mainly in women, the elderly and pregnant women. The main etiological agent is E. coli, especially in Community infections. Antimicrobial therapy is performed extensively with the Quinolones and resistance emergence is striking and upward. It is known E. coli resistance mechanisms are related with QRDR (Quinolone Resistance Region Determining) mutations in gyrA and parC genes that are the drug targets. These genes encode respectively DNA Girase and Topoisomerase IV subunits responsible for DNA supercoiling and DNA decatenating. Another resistance mechanism that come from plasmids is qnr genes encoding a protein that can protect DNA girase and Topoisomerase IV. Another plasmidial gene is aac(6´)-Ib-cr encoding an aminoglycoside acetyltransferase that can inactivate ciprofloxacin and norfloxacin. This study analyzed 58 E. coli lactose negative samples from urine, in order to draw a profile of resistance in this population from the region of Ponta Grossa and analyze the resistance mechanisms involved. There was a high resistance rate of 48%, among which 79% were resistant to all quinolones tested: nalidixic acid, ciprofloxacin, norfloxacin and ofloxacin. There was also a direct relationship between the biotypes 971 and resistance in contrast to biotype 981 and sensitivity. Therefor Quinolones are not recommended in infection due to E. coli biotype 971. On molecular analysis on the multiresistant samples mutations with aminoacid substitution were found in three positions: the gyrA gene at codons 83 and 87 and in the parC gene at codon 80 in three strains and at codon 84 on a single strain. Mutations only at gyrA gene appeared for a sample resistant just to nalidixic acid. These facts are according to the theory of mutations accumulation causing increased resistance to quinolones. Plasmidial genes qnr and aac(6´)-Ib-cr were not found in these strains. / As infecções do trato urinário mostram uma elevada ocorrência na população, especialmente em mulheres, gestantes e idosos. O principal agente etiológico é a E. coli, principalmente nas infecções de origem comunitária. A terapia com antimicrobianos é realizada extensivamente com Quinolonas e o aparecimento de resistência é marcante e ascendente. Os principais mecanismos de resistência conhecidos em E. coli estão relacionados a mutações nas QRDR (Região Determinante de Resistênbcia a Quinolonas) dos genes gyrA e parC, codificadores de subunidades das enzimas DNA Girase e Topoisomerase IV, respectivamente. Essas enzimas, que são os alvos de ação das Quinolonas, são responsáveis pelo superespiralamento e decatenação do DNA. Os genes plasmidiais que também estão envolvidos nos mecanismos de resistência são os qnr que codificam proteínas protetoras das enzimas alvo e o aac(6´)-Ib-cr, que codifica uma enzima aminoglicosídio acetiltransferase capaz de inativar a ciprofloxacina e norfloxacina. Neste trabalho foram analisadas 58 amostras de E. coli lactose negativas provenientes de urina, a fim de se traçar um perfil de resistência nessa população da região de Ponta Grossa e analisar os mecanismos de resistência envolvidos. Observou-se uma elevada taxa de resistência de 48%, dentre as quais 79% foram resistentes a todas as quinolonas testadas: ácido nalidíxico, ciprofloxacina, norfloxacina e ofloxacina. Pode-se observar também uma relação direta entre os biótipos 971 e uma maior resistência e o biótipo 981 e uma maior sensibilidade, motivo pelo qual não seria recomendável o uso de Quinolonas para o tratamento de infecções causadas por E. coli biótipo 971. Pela análise molecular, para as amostras multirresistentes, foram encontradas mutações com substituição de aminoácido nos genes gyrA nos códons 83 e 87 e no gene parC uma única mutação, sendo essa no códon 80 para três cepas e no códon 84 para uma cepa. Mutações apenas no gene gyrA apareceram para amostras resistentes somente ao ácido nalidíxico. Esses dados estão de acordo com a teoria de que o acúmulo de mutações leva a um aumento da resistência às quinolonas. Os genes plasmidiais qnr e aac(6´)-Ib-cr não foram encontrados.

Dentre as espécies do gênero Yersinia, Yersinia enterocolitica é a espécie mais prevalente como causa de doença em humanos e animais. Y. enterocolitica é dividida em seis biotipos. Os biotipos 1B, 2, 3, 4 e 5 compreendem linhagens associadas à doença em humanos e animais, enquanto o biotipo 1A consiste de linhagens consideradas não patogênicas. Apesar de Y. enterocolitica biotipo 2 ser de importância clínica, há uma escassez de estudos no país, o que dificulta avaliar o envolvimento dessa bactéria como causa de doença em humanos e em animais, bem como, determinar o impacto de sua presença no meio-ambiente. O objetivo deste trabalho foi investigar o potencial patogênico, determinar o perfil de suscetibilidade a antimicrobianos e verificar a diversidade genotípica de linhagens de Y. enterocolitica biotipo 2 isoladas no Brasil. Foram estudadas 40 linhagens de Y. enterocolitica biotipo 2, isoladas de humanos (5), ambiente (34) e animal (1), entre os anos de 1979 e 1998. Ademais, nas análises filogenéticas, foram acrescidas 26 linhagens de Y. enterocolitica pertencentes aos outros biotipos, com o intuito de comparar as linhagens de Y. enterocolitica biotipo 2 aos biotipos 1A, 1B, 3, 4 e 5. As linhagens de humanos e animal foram sensíveis a todos os 14 antimicrobianos testados. Dentre as 34 linhagens de ambiente, sete (20,6%) foram resistentes a um ou dois antimicrobianos, sendo esses, amicacina, cefoxitina, gentamicina, e sulfametoxazol - trimetoprima. Todas as linhagens apresentaram os genes inv, ail, ystA, hreP, tccC e myfA. Os genes fepD e fes foram detectados em 39 (97,5%) linhagens, o gene virF foi encontrado em três (7,5%) linhagens, os genes ystB e fepA não foram detectados em nenhuma linhagem. Todas as linhagens apresentaram comportamento relacionado à virulência frente aos testes fenotípicos de atividade da pirazinamidase, hidrólise da esculina e fermentação da salicina. O dendrograma de similaridade genética de Enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) agrupou as linhagens de Y. enterocolitica biotipo 2 em cinco grupos denominados A, B, C, D e E. Todas as linhagens, com exceção de duas, apresentaram similaridade genética superior a 88,3%. O dendrograma de similaridade genética de Pulsed field gel electrophoresis (PFGE) agrupou as linhagens de Y. enterocolitica biotipo 2 em três grupos denominados I, J e K. A maioria das linhagens (72,5%) apresentou similaridade ii genética superior a 78,3%. O dendrograma de similaridade genética de Multilocus variable number tandem repeat analysis (MLVA) agrupou as linhagens de Y. enterocolitica biotipo 2 em dois grupos denominados O e P com similaridade genética superior a 37,7%. Pode-se concluir que o potencial patogênico das linhagens de Y. enterocolitica biotipo 2 foi evidenciado pela prevalência da maioria dos marcadores de virulência, bem como, pelo comportamento relacionado à virulência frente aos testes fenotípicos pesquisados. Algumas linhagens apresentaram-se resistentes a antimicrobianos de primeira escolha no tratamento de yersiniose, o que pode acarretar em falha terapêutica. Os resultados de ERIC-PCR e PFGE mostraram a alta similaridade entre as linhagens de Y. enterocolitica biotipo 2, sugerindo que as mesmas pouco se diferenciaram ao longo dos 19 anos e que possivelmente o meio ambiente tem sido uma fonte de contaminação para humanos e animais no Brasil. A técnica de MLVA agrupou as linhagens de Y. enterocolitica biotipo 2 quanto à sua origem e a técnica de ERIC-PCR agrupou as linhagens de Y. enterocolitica biotipos 1A, 1B, 2, 3, 4, e 5 quanto às diferentes patogenicidades características de cada biotipo. / Among the species of the genus Yersinia, Yersinia enterocolitica is the most prevalent species that cause illness in humans and animals. Y. enterocolitica is divided into six biotypes. Biotypes 1B, 2, 3, 4 e 5 comprise strains associated to illness in humans and animals, while biotype 1A comprise strains considered nonpathogenic. Despite of the fact that Y. enterocolitica biotype 2 is of clinical importance, there is a paucity of studies in this country, which makes difficult to assess the involvement of this bacteria as a cause of illness in humans and animals, as well as to determine the impact of its presence in the environment. The aim of this work was to investigate the pathogenic potential, to determine the antimicrobial resistance profile and to verify the genetic diversity of Y. enterocolitica biotype 2 strains isolated in Brazil. Forty strains of Y. enterocolitica biotype 2 isolated from humans (5), environment (34) and animal (1), between 1979 and 1998 were studied. Besides, in the phylogenetic analyzes it was added 26 Y. enterocolitica strains belonging to the other biotypes, in order to compare the Y. enterocolitica biotype 2 strains to biotypes 1A, 1B, 3, 4 e 5. Humans and animals strains showed susceptibility to all 14 antibiotics tested. Among the 34 environment strains, seven (20.6%) were resistant to one or two antibiotics used such as amikacin, cefoxitin, gentamicin and sulfamethoxazole-trimethoprim. All the strains presented the genes inv, ail, ystA, hreP, tccC and myfA. Genes fepD and fes were detected in 39 (97.5%) strains, virF was found in three (7.5%) strains, and ystB and fepA were not detected in any strains. All the strains exhibited behavior related to virulence against the phenotypic tests of pyrazinamidase activity, esculin hydrolysis and salicin fermentation. The dendrogram of genetic similarity of Enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) grouped the Y. enterocolitica biotype 2 strains in five groups, designated A, B, C, D and E. All the strains, except two, showed a genetic similarity of more than 88.3%. The dendrogram of genetic similarity of Pulsed field gel electrophoresis (PFGE) grouped the Y. enterocolitica biotype 2 strains in three groups, designated I, J and K. The majority of the strains (72.5%) showed a genetic similarity of more than 78.3%. The dendrogram of genetic similarity of Multilocus variable number tandem repeat analysis (MLVA) grouped the Y. enterocolitica iv biotype 2 strains in two groups, designated O and P with a genetic similarity of more than 37.7%. It is possible to conclude that the pathogenic potential of the Y. enterocolitica biotype 2 strains was highlighted by the prevalence of the majority of the virulence markers searched, as well as by the behavior related to virulence against the phenotypic tests. Some strains were resistant to antimicrobials that are the first choice for yersiniosis treatment, which can result in therapeutic failure. The results of ERIC-PCR and PFGE showed a high genetic similarity between the Y. enterocolitica biotype 2 strains, suggesting that the strains differed little over 19 years, and that the environment has been possibly a source of humans and animals infections in Brazil. The MLVA technique grouped the Y. enterocolitica biotype 2 strains according their origins, and the ERIC-PCR technique grouped the Y. enterocolitica biotypes 1A, 1B, 2, 3, 4 and 5 strains according to the different pathogenicity characteristics of each biotype.

The lyssavirus genus of the Rhabdovirus family is divided into seven genotypes. Genotype 3, Mokola virus, has only been found on the African continent, and has been reported to infect rodents, cats, dogs and humans. The first Mokola virus identification in South Africa was made in 1970, on the east coast of the KwaZulu-Natal province. After 25 years, Mokola virus was again identified in three cats, 650 km south-west of the previous isolation. In 1997 two more Mokola infections were identified in Pinetown, only about 23 km south-west of the 1970 isolation. Phylogenetic analysis of the nucleic acid sequences of the nucleoprotein gene region of the Mokola genome, indicated that the Mokola viruses from the same geographical region were more closely related, irrespective of the time of isolation. The identification of these two distinct clusters of Mokola in South Africa leads i us to believe that this virus is more widespread than previously thought, but that the reservoir host species remains to be identified. Genotype 1 in the Rhabdovirus family, rabies virus, is found on all continents, except Australia, New Zealand, Papua New Guinea, Japan, Hawaii, Taiwan, United Kingdom, Ireland, etc. An ongoing rabies enzootic in southern Africa is associated with two genetically distinct groups of viruses, called the canid biotype (infecting carnivores of the family Canidae) and the viverrid biotype (infecting carnivores of the subfamily Viverrinae). We identified the first cases of spillover of canid biotype virus into viverrid hosts, using monoclonal antibody and nucleic acid sequence analysis. Genetic analysis of the G-L intergenic region of the rabies virus genome, showed that these spillover events do not bring about any significant change on this part of the virus genome. All of these spillover isolates maintained a typical canid virus phylogeny. Rabies viruses associated with the family Viverridae form a highly diverse group of viruses, which can be divided into four distinct phylogenetic groups, each associated with a specific geographical area in South Africa. The canid biotype of rabies virus is divided into three specific groups, based on geographic location and the associated reservoir species, namely KwaZulu-Natal province (with domestic dogs as its main vector), the western parts of South Africa (bat-eared foxes) and the northern parts of South Africa (black-backed jackals). In order to determine the degree of genetic change in the virus over a period of time, we identified two endemic canid rabies regions (KwaZulu-Natal and the northern parts of South Africa) and analysed the nucleic acid sequence variation 0f the viruses over 15 years. Phylogenetic analysis of the variable G-L intergenic region of t e virus genome indicated that the canid rabies biotype changed less than 1% over the period studied. This implies that the highly diverse viverrid biotype has been circulating in the southern African wildlife for a very long time. In order to obtain a faster, more economical, and reliable method for rabies virus biotype identification, a competitive, hemi-nested PCR assay was developed. In a single tube, two biotype specific oligonucleotides (developed by Jaftha, 1997), and a common downstream primer were -used in the biotype specific, second round amplification. The specific virus biotypes were identified on the basis of specific amplicon sizes for each biotype. A third biotype specific primer was designed to target a region of the Nucleoprotein gene, this primer was used in a second round hemi-nested reaction. Despite having been designed to specifically amplify canid biotype viruses, this primer amplified all rabies biotypes non¬specifically. We conclude that the nucleoprotein genes are too conserved to make this part of the genome a good target for a biotype-specific PCR diagnostic assay. / Dissertation (MSc (Agric) Microbiology)--University of Pretoria, 1997. / Microbiology and Plant Pathology / unrestricted

<p>Within the last few decades, swine breeding programs have been refined to include pork quality and novel carcass traits alongside growth, feed efficiency, and carcass leanness in the selection programs for terminal sire lines with a goal to produce high quality and efficient pork product for consumers. In order to accurately select for multiple traits at once, it becomes imperative to explore their genetic and biological architecture. The genetic architecture of traits can be explored through the estimation of genetic parameters, genome-wide association studies (GWAS), gene networks and metabolic pathways. An alternative approach to explore the genetic and biological connection between traits is based on principal component analysis (PCA), which generates novel “pseudo-phenotypes” and biological types (biotypes). In this context, the main objective of this thesis was to understand the genetic and biological relationship between three growth, eight conventional carcass, 10 pork quality, and 18 novel carcass traits included in two studies. The phenotypic data set included 2,583 records from female Duroc pigs from a terminal sire line. The pedigree file contained 193,764 animals and the genotype file included 21,344 animals with 35,651 single nucleotide polymorphisms (SNPs). The results of the first study indicate that genetic progress can be achieved for all 39 traits. In general, the heritability estimates were moderate, while most genetic correlations were generally moderate to high and favorable. Some antagonisms were observed but those genetic correlations were low to moderate in nature. Thus, these relationships can be considered when developing selection indexes. The second study showed that there are strong links between traits through their principal components (PCs). The main PCs identified are linked to biotypes related to growth, muscle and fat deposition, pork color, and body composition. The PCs were also used as pseudo-phenotypes in the GWAS analysis, which identified important candidate genes and metabolic pathways linked to each biotype. All of this evidence links valuable variables such as belly, color, marbling, and leanness traits. Our findings greatly contribute to the optimization of genetic and genomic selection for the inclusion of valuable and novel traits to improve productive efficiency, novel carcass, and meat quality traits in terminal sire lines.<br></p><p></p>

In field experiments, managing for earliness through planting date and varietal maturity reduced tarnished plant bug, Lygus lineolaris (Palisot de Beauvois), densities, insecticide applications, and yield loss. A second experiment highlighted the importance of timely insecticide applications for managing tarnished plant bugs. Differences in fitness parameters were observed between tarnished plant bug populations collected from the Hills and Delta regions of Mississippi. Populations from the Delta region laid more eggs and produced more viable offspring than populations from the Hills. Populations from the Delta reared on cotton developed significantly faster to each life stage than those reared on diet or populations from the Hills region. Overall, tarnished plant bugs survived significantly better on diet than on cotton. Results from these experiments will be important for improving IPM practices for tarnished plant bugs in Mississippi cotton.

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