Thesis (doctoral)--Universiteit van Amsterdam, 1989. / Summary in Dutch. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (doctoral)--Lund University, 1997. / Added t.p. with thesis statement inserted.

Made available in DSpace on 2014-08-26T17:15:20Z (GMT). No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) 145.pdf: 1867505 bytes, checksum: 87dab03551d06be6de32b042de770c29 (MD5) Previous issue date: 2005 / Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde / O presente estudo incluiu 235 cepas, procedentes dos estados de Santa Catarina (SC), Rio de Janeiro (RJ) e Pemambuco (PE). Verificamos que o Hib antes da vacinação predominava em mais de 90 por cento dos casos, sendo os outros tipos sorológicos raros. Após a vacinação, cai o tipo,b, crescem os tipos não b, em meningites, septicemias e infecções respiratórias. Os biotipos I e II foram os mais isolados. Houve variação da sensibilidade aos antimicrobianos testados (ampicilina, amoxicilina-ácido clavulânico, ceftriaxona, rifampicina, cloranfenicol e sulfametoxazol-trimetoprim), sobretudo na associação SMX- TMP cuja resistência dos Hi aumentou de 32,6 por cento para 65,8 por cento entre os dois períodos; enquanto se mantiveram taxas semelhantes de resistência para a ampicilina (17,5 por cento e 15,8 por cento) e a presença de cepas produtoras de p-lactamase; a sensibilidade à amoxicilina-ácido clavulânico e ceftriaxona foi de 100 por cento. Alteração na resistência ao cloranfenicol de 19,4 para 12,5 por cento e da rifampicina de 8,2 para 9,7 por cento.A tipagem sorológica realizada pelo método da SAL, teve bons resultados (96,2 por cento) e a molecular, pela PCR, diferenciou as cepas NT das capsuladas b -.Embora a estrutura populacional dos Hi tenha sido inicialmente descrita como clonal, os resultados obtidos através da técnica de ERIC- PCR, revelaram diversidade genética nas cepas estudadas. As do sorotipo b, revelaram 4 clones com diferentes características epidemiológicas. A diversidade genética foi maior nas Hinb e HiNT. Dos estados estudados, PE teve a mais alta diversidade genética (6 clones em 15 cepas) seguido do RJ (3 clones em 23 cepas) e SC (2 clones em 13 cepas). Concluímos, portanto, que há necessidade do monitoramento das cepas circulantes de Hi no país observando as possíveis alterações na prevalência dos sorotipos atuais e a real estimativa do impacto da vacina contra o Hib atualmente utilizada no Brasil. / The bacterial genus Haemophilus is included in the family Pasteurellaceae and the mostimportant specie causing infectious diseases in humans is the Hi. It shows capsularserotypes (a-f) as well as NT strains. A great number of different acute infectious diseasesare caused by this organism, especially affecting the CNS, as well as RD, occurringfrequently in children, especially with the Hib. Since 1988 disease associated with Hib hasbeen vaccine-preventable, by means of the first conjugate vaccine showing great efficacy.The vaccine is composed by the PRP and a carrier protein. However, the Hib is stillconsidered one of the most important human pathogens, which leaded us to developdifferent research lines, with isolates from different source of infections, after theintroduction of the conjugate vaccine against Hib by PNI/MS in 1999. We used in ourstudy 235 Hi isolates, from the brazilian states of Santa Catarina, Rio de Janeiro andPernambuco. Our results, show that the rate of type b infections before vaccination wasmore than 90%, whereas other serological types were rare. After the advent of the vaccine,we observe a decrease of type b and an increase of non-b isolation. Biotypes I and II arestill the most frequently isolated. The antimicrobial susceptibility found against ampicillin,amoxicillin-clavulanate, ceftriaxone, rifampicin, chloranphenicol and TMP-SMX, showed arecent increasing resistance against antibiotics commonly used to treat RD, especiallyamong the ones administrated by oral route, showing a resistance rate from 32.6% to 65.8%between the two periods studied. Ampicillin, showed resistance rates of 17.5% and 15.8%in the two periods studied and the presence of â-lactamase producing strains. Strainsevaluated against amoxicillin/clavulanate and ceftriaxone, were 100% sensible.Chloranphenicol showed a decrease in the resistance rates of the strains isolated during thepost-vaccination period, while rifampicin, showed an increase in the resistance rate from8.2 to 9.7%. The seroaglutination method used for capsular typing, showed good results,however, mutant b capsulated strains (b-), were only detected by the PCR, using specificprimers for the capsule region. The population structure of Hi which has been described asclonal, revealed a great genetic diversity by ERIC-PCR. Therefore we can conclude thatthere is a need of systematic tracking of circulating Hi strains in the country, continuouslywatching possible epidemiological changes and the evaluation of the impact of the vaccineagainst Hib used in Brazil to date.

Made available in DSpace on 2015-02-27T17:39:02Z (GMT). No. of bitstreams: 2 81.pdf: 872019 bytes, checksum: b8e894b7b58911018e4a590c0266cbe1 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2010 / Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde / A bactéria Haemophilus da espécie influenzae, pode causar no homem diversas infecções, invasivas ou não. O H. influenzae do tipo b (Hib), antes da introdução da vacina conjugada contra Hib, associava-se a infecções como epiglotite, artrite séptica, bacteriemia, pneumonia septicemia e meningite, principalmente em crianças. As outras espécies tipáveis (a,c,d,e,f) e não tipáveis estavam associadas a infecções do trato respiratório adquiridas na comunidade. Após a introdução dessa vacina, houve redução expressiva das doenças causadas pelo Hib em diversas partes do mundo, inclusive no Brasil. Atualmente, outros tipos da espécie H. influenzae (Hi) que não o Hib estão sendo isoladas não somente em crianças, causando infecções graves. O ressurgimento de casos por Hib, em crianças vacinadas, tem sido reportado como possível falha vacinal. Esses fatos preocupam a Saúde Pública no mundo, demonstrando que mudanças epidemiológicas podem estar ocorrendo devido à seleção de cepas após a introdução da vacina. Neste estudo, mudanças genéticas na região capsular do Hi foram encontradas em cepas isoladas nos períodos pré e pós-vacinal de três estados brasileiros. Através do estudo de susceptibilidade foram encontradas cepas produtoras de β-lactamase (BLA+) e não produtoras resistentes a ampicilina (BLNAR). A análise de redução da susceptibilidade às quinolonas foi avaliada utilizando-se o ácido nalidíxico como marcador, confrontando-se a resistência deste antibiótico com os marcadores moleculares das regiões determinantes de resistência a quinolonas (QRDRs) através do sequenciamento dos genes gyrA e parC que codificam a síntese das proteínas GyrA e ParC. Os resultados obtidos demonstraram que o ácido nalidíxico é o antibiótico mais indicado desse grupo para detectar a redução da sensibilidade. / A bactéria Haemophilus da espécie influenzae, pode causar no homem diversas infecções, invasivas ou não. O H. influenzae do tipo b (Hib), antes da introdução da vacina conjugada contra Hib, associava-se a infecções como epiglotite, artrite séptica, bacteriemia, pneumonia septicemia e meningite, principalmente em crianças. As outras espécies tipáveis (a,c,d,e,f) e não tipáveis estavam associadas a infecções do trato respiratório adquiridas na comunidade. Após a introdução dessa vacina, houve redução expressiva das doenças causadas pelo Hib em diversas partes do mundo, inclusive no Brasil. Atualmente, outros tipos da espécie H. influenzae (Hi) que não o Hib estão sendo isoladas não somente em crianças, causando infecções graves. O ressurgimento de casos por Hib, em crianças vacinadas, tem sido reportado como possível falha vacinal. Esses fatos preocupam a Saúde Pública no mundo, demonstrando que mudanças epidemiológicas podem estar ocorrendo devido à seleção de cepas após a introdução da vacina. Neste estudo, mudanças genéticas na região capsular do Hi foram encontradas em cepas isoladas nos períodos pré e pós-vacinal de três estados brasileiros. Através do estudo de susceptibilidade foram encontradas cepas produtoras de β-lactamase (BLA+) e não produtoras resistentes a ampicilina (BLNAR). A análise de redução da susceptibilidade às quinolonas foi avaliada utilizando-se o ácido nalidíxico como marcador, confrontando-se a resistência deste antibiótico com os marcadores moleculares das regiões determinantes de resistência a quinolonas (QRDRs) através do sequenciamento dos genes gyrA e parC que codificam a síntese das proteínas GyrA e ParC. Os resultados obtidos demonstraram que o ácido nalidíxico é o antibiótico mais indicado desse grupo para detectar a redução da sensibilidade. O presente estudo mostra a necessidade de que os laboratórios de rotina e de pesquisa avaliem e desenvolvam métodos simples e rápidos, capazes de indicar as possíveis mudanças capsulares e o surgimento de cepas resistentes auxiliando a Vigilância Epidemiológica e a Vigilância Sanitária.

Made available in DSpace on 2015-07-08T12:28:20Z (GMT). No. of bitstreams: 2 5.pdf: 1093384 bytes, checksum: 9432b61bcb4cfac37f05a1600b8f2fae (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2013 / Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde / Haemophilus influenzae pode ser encontrado, normalmente, na microbiota do trato respiratório, do trato gênito-urinário e da cavidade oral. Porém, essa espécie inclui um dos mais importantes patógenos bacterianos em infecções principalmente pediátricas. As cepas de Hi podem ser capsuladas, variando de a-f, ou não capsuladas (não tipáveis - NT). O tipo capsular b foi o mais frequente em infecções graves infantis até a utilização da vacina conjugada contra Hib, sendo ainda considerado patogênico. No Brasil, essa vacina foi introduzida no Programa Nacional de Imunização do Ministério da Saúde em agosto de 1999, e como em outros países, promoveu uma acentuada diminuição em doenças causadas por esse agente. No entanto, estudos realizados na era pós-vacinal têm mostrado que a incidência de doenças invasivas causadas por H. influenzae não b e NT têm aumentado, inclusive no Brasil. O objetivo desse trabalho foi obter informações sobre as cepas de Hi circulantes no município do Rio de Janeiro. Utilizou-se 96 amostras de quadros infeciosos (46 invasivas e 50 não invasivas), isoladas no período pós-vacinal (2000-2012). Em relação à idade, dos 54 pacientes que tiveram esse dado, 32 tiveram doenças invasivas e 22 não invasivas. Em doenças invasivas, houve o predomínio de crianças < 5 anos. Enquanto que nas não invasivas os adultos > 70 anos predominaram. Entre as cepas obtidas, 15 foram capsuladas e 81 não capsuladas. A maioria das cepas capsuladas foi proveniente de sítios invasivos, cuja faixa etária predominante foi de < 5 anos. O tipo capsular mais isolado foi o b, seguido do a e f. As cepas Hib, predominaram no início do período de estudo, enquanto os outros sorotipos predominaram no final. No presente trabalho, não encontramos cepas mutantes deficientes de cápsula tanto Hib-, quanto Hia- / Haemophilus influenzae can be found usually in the microbiota of the respiratory tract, genitourinary tract and oral cavity. However, this species includes one of the most important bacterial pathogens mainly in pediatric infections. The strains can be capsulated Hi, (serotypes a-f), or not capsulated (nontypeable - NT). The type b capsular was the most frequent serious infection in children until the use of conjugate vaccine against Hib and is still considered pathogenic. In Brazil, this vaccine was introduced in the National Immunization Program of the Ministry of Health in August 1999, and as in other countries, promoted a significant decrease of disease caused by this agent. However, studies during the post-vaccine period have shown that the incidence of invasive disease caused by H. influenzae b and NT have increased, including in Brazil. The aim of this study was to obtain information on the Hi strains circulating in the municipality of Rio de Janeiro. We used 96 samples (46 invasive and 50 noninvasive), isolated during the post-vaccination period (2000-2012). Concerning the age of the 54 patients who had this data, 32 had invasive and 22 noninvasive disease. In invasive disease, there was a predominance of children <5 years. While in the noninvasive group, adults > 70 years predominated. Among the strains obtained, 15 were capsulated and 81 non-capsulated. Most capsulated strains originated from invasive sites whose predominant age group was <5 years. The most frequent capsular type was b, followed by a and f. Hib strains predominated at the beginning of the study period, while the other serotypes prevailed in the end. In this study, we did not found mutant strains deficient in both capsule Hib- and Hia-. NT strains accounted for the vast majority of isolates in this study, 32 strains isolated from invasive sites and 49 sites noninvasive, and were obtained from patients of all age groups. The capsulated strains were predominantly biotype I and II, while non-typeable strains were most II and III. In this study, only the NT strains were resistant to two drugs: ampicillin and trimethoprim - sulfamethoxazole. These were mostly non-invasive. Thus, none of the capsulated isolates were resistant. The PFGE patterns for the 96 strains were quite different, however eight NT strains belonged to the same genotype. Capsulated strains of the same serotype were similar, getting most isolates grouped in the same cluster. We therefore conclude that it is necessary to monitor the Hi strains circulating in Rio de Janeiro, because of the geographic and economic importance of this municipality. Such conduct should be extended to the whole country in order to understand the possible changes of serotypes today, which will certainly guide for the design of new vaccines, improvement of existing ones and the use of antibiotics, resulting in a public health impact.

No description available.

H. influenzae ist ein fakultativ anaerobes, Gram-negatives Bakterium und wird in die Familie der Pasteurellacaea eingeordnet. Das Bakterium zeigt Auxotrophien für Hämin unter aeroben Bedingungen und für Nikotinamid-Adenin-Dinukleotid (NAD) bei aeroben wie anaeroben Wachstum. Es können zwei Unterarten unterschieden werden: die bekapselten Stämme, die systemische Erkrankungen hervorrufen und die unbekapselten bzw. nicht-typisierbaren Stämme, die für Oberflächeninfektionen verantwortlich sind. Da bei H. influenzae bisher nur wenig über die NAD-Aufnahme bekannt war, sollten in dieser Arbeit Proteine identifiziert und charakterisiert werden, die an der NAD-Aufnahme beteiligt sind. Das Außenmembranprotein e(P4), kodiert von dem hel-Gen, wurde als eine Komponente des Häminaufnahmesystems beschrieben. In dieser Arbeit wurde eine hel-Deletionsmutante hergestellt, anhand der nachgewiesen wurde, daß e(P4) als saure Phosphatase nicht an der Hämin-, sondern an der NAD-Aufnahme beteiligt ist. Mit Hilfe von verschiedenen Phosphatase-Assays und dem Malat-Enzym-Assay konnten NADP und Nikotinamid-Mononukleotid (NMN) als physiologisch relevante Substrate identifiziert werden. Die biologische Relevanz von e(P4) für die NAD-Aufnahme wurde durch Mutantenanalyse in Wachstumstests und Transport-Assays nachgewiesen. Es wurde gezeigt, daß die hel-Deletionsmutante mit NAD und NMN ein signifikantes Wachstumsdefizit hatte und nicht fähig war, beide Nikotinamid-Nukleotid-Quellen aufzunehmen, während die Nutzung von Nikotinamid-Ribosyl (NR) keinen Unterschied zum Wildtyp aufwies. Um die Frage zu klären, ob die Lokalisation von e(P4) als Lipoprotein an der Außenmembran wichtig für die NMN-Spaltung ist, wurden zwei Mutanten hergestellt, bei denen im hel-Gen der Lipidanker Cystein durch ein Glycin ausgetauscht wurde, um das Protein im Periplasma zu exprimieren. Die Periplasma-Extrakte dieser Cystein-Mutanten zeigten in Phosphatase-Assays keine Aktivität. Um zu untersuchen, ob die Phosphatase-Aktivität die einzige für die NAD-Aufnahme relevante Funktion von e(P4) ist, wurden Phosphatase-Mutanten hergestellt und charakterisiert. Sie exprimierten ein mutiertes e(P4)-Protein, das durch einen Aminosäureaustausch die Phosphatase-Aktivität verloren hatte. Es zeigte sich, daß die Phänotypen der Phosphatase-Mutanten in Bezug auf die Fähigkeit NMN zu spalten, NAD und NMN aufzunehmen und als Faktor V-Quelle zum Wachstum zu nutzen, exakt mit den Phänotypen der Deletionsmutante korrelierten. Es konnten daher keine weiteren Funktionen von e(P4) festgestellt werden. Das periplasmatische Protein NadN wurde als Pyrophosphatase beschrieben, die fähig ist, NAD zu NMN zu hydrolysieren. Weiter wurde eine 5´-Nukleotidase-Aktivität nachgewiesen, mit der NadN NMN zu NR dephosphorylieren kann. Die Relevanz von NadN für die NAD-Aufnahme wurde anhand einer nadN-Knockout-Mutante untersucht. In Wachstumskurven und Transport-Assays zeigte sich, daß die nadN-Mutante nicht fähig war, NAD aufzunehmen und zum Wachstum zu verwenden. Auch die Nutzung von NMN war bei der Mutante eingeschränkt, während mit NR als Faktor V-Substrat kein Unterschied zum Stamm Rd erkennbar war. Durch die Charakterisierung einer hel nadN-Doppelmutante konnte nachgewiesen werden, daß keine weiteren Enzyme außer e(P4) und NadN an der Prozessierung von NAD zu NR beteiligt sind. Es zeigte sich auch, daß nur NR über einen putativen Transporter ins Zytoplasma aufgenommen wird. Das Protein OmpP2 stellt ein Hauptporin der äußeren Membran dar. Durch eine ompP2-Deletionsmutante konnte mit Hilfe von Wachstumskurven und Transport-Assays nachgewiesen werden, daß NAD, NMN und NR durch diese Pore ins Periplasma diffundieren. / H. influenzae is a facultativ anaerobic, Gram-negative bacterium and belongs to the familiy of Pasteurellaceae. This bacterium shows auxotrophies for hemin under aerobic conditions and for nicotinamid adenine dinucleotide (NAD) at aerobic and anaerobic growth. Two subspecies are distinguishable: the encapsulated strains, the causative agents for systemic and invasive diseases and the unencapsulated or nontypeable strains, responsible for inflammation of the respiratory tract. In H. influenzae little is known about the utilization of NAD, therefore this work was aimed to identify and characterize gene products which are involved in the uptake of NAD. The outer membrane protein e(P4), encoded by the gene hel, was described as a component of the hemin uptake system. In this work a hel deletion mutant was constructed to proof that the acid phosphatase e(P4) is not involved in the uptake of hemin but in the uptake of NAD. With the aid of different phosphatase assays and the maleic enzyme assay NADP and NMN could be identified as the physiological relevant substrates. The biological relevance of e(P4) was proofed by mutant analysis in growth tests and transport assays. It was shown that the hel deletion mutant had a significant growth deficiency with NAD and NMN and was not able to take up both nicotinamide nucleotide sources, whereas the utilization of nicotinamide ribosyl (NR) showed no difference compared to the wildtype. To address the question, wether the localization of the lipoprotein e(P4) at the outer membrane is important for the hydrolysis of NMN, two mutants were constructed which had a Cystein-Glycin replacement in the lipid anchor of the hel gene to express the protein in the periplasm. The periplasm extracts of these Cystein mutants showed no activity in phosphatase assays. To investigate wether the phosphatase acitivty of e(P4) is the only relevant function for the uptake of NAD, phosphatase mutants were constructed and characterized. They expressed a mutated e(P4) protein which had lost the phosphatase activity by an amino acid replacement. The phenotypes of the phosphatase mutants correlated exactly with the phenotype of the deletion mutant concerning the ability to cleave NMN, to take up NAD and NMN and to use them as factor V sources. The periplasmic protein NadN was described as a pyrophosphatase which is able to hydrolase NAD to NMN. Further a 5´-nucleotidase function was identified enabling NadN to dephosphorylate NMN to NR. The relevance of NadN for the utilization of NAD was investigated with a nadN knockout mutant. Growth curves and transport assays revealed that the nadN mutant was not able to take up NAD and to use it for growth. The utilization of NMN was reduced, whereas the utilization of NR showed no difference compared to the wildtype. By characterizing a hel nadN double mutant it was shown that no further enzymes than e(P4) and NadN are involved in the processing of NAD to NR and that only NR is taken up into the cytoplasm through a putativ transporter. The protein OmpP2 is the major porin of the outer membrane. Growth curves and transport assays with an ompP2 deletion mutant revealed that NAD, NMN and NR diffuse through this porin into the periplasm.

En el presente estudio se diseñó una técnica molecular alternativa para la identificación de Haemophilus paragallinarum, agente causal de la Coriza Infecciosa, basada en el polimorfismo en longitud de los fragmentos de restricción de los genes ribosómicos 16S amplificados por la reacción en cadena de la polimerasa (RFLP-PCR). Esta metodología fue propuesta debido a que las técnicas, HPG-1 y HPG-2, las cuales vienen siendo utilizadas en la identificación de este patógeno, mostraron baja sensibilidad y reacción cruzada con Escherichia coli, respectivamente. En la técnica HPG-2 se encontró dos productos de amplificación entre 450 y 500 pb confundiéndose con el producto de 500 pb que se obtiene con H. paragallinarum. Para la estandarización de la técnica molecular propuesta, se utilizaron las cepas de referencia ATCC 29545, B y C de H. paragallinarum, y como controles negativos a Ornithobacterium rhinotracheale, Pasteurella spp., microorganismos con características morfológicas, bioquímicas y cuadros clínicos similares a H. paragallinarum; y E. coli, esta última, asociada en la Coriza Infecciosa complicada. Se utilizaron cebadores universales para amplificar los genes ribosómicos 16S con los que se obtuvo un fragmento de 1500 pb para todas las cepas, éstos fueron cortados con enzimas de restricción seleccionadas mediante el análisis de las secuencias de los genes ribosómicos 16S depositadas en el GenBank (banco de genes). Con el RFLP-PCR se obtuvo una mayor especificidad y sensibilidad ya que se identificaron perfiles genéticos específicos para H. paragallinarum y diferentes a los de O. rhinotracheale, Pasteurella spp y E. coli. La técnica diseñada fue utilizada en la identificación de 19 cepas aisladas de muestras clínicas de senos infraorbitarios y cornetes nasales de gallinas reproductoras, de postura y pollos de carne de diversas zonas del Perú obteniendo resultados reproducibles y demostrando la validez de esta técnica y su uso para una rápida identificación de microorganismos implicados en infecciones respiratorias en aves comerciales. Palabras claves: Haemophilus paragallinarum, Ornithobacterium rhinotracheale, Coriza Infecciosa, genes ribosómicos 16S, RFLP-PCR. / --- In the present study, a technical molecular alternative for the identification of Haemophilus paragallinarum, causal agent of the Infectious Coryza, based on the restriction fragments length polymorphism of the 16S ribosomal genes amplified by the chain reaction polymerase (RFLP-PCR) was designed. This methodology was proposed due to the fact that the HPG-1 and HPG-2 techniques, which have been used for the identification of this pathogen, showed low sensitivity and crossed reaction with Escherichia coli respectively. In the HPG-2 technique, two amplification products between 450 and 500 bp were obtained, similar with the 500 bp product obtained with H. paragallinarum. For the standardization of the proposed molecular technique, we used the ATCC 29545 reference strain and the B and C strains of H. paragallinarum, as well as Ornithobacterium rhinotracheale, Pasteurella spp., microorganisms with similar morphological, biochemical characteristics and clinical signs to those of H. paragallinarum, and E. coli, bacterium associated in the complicated Infectious Coryza, as negative controls. Universal primers for amplifying the 16S ribosomal genes were used, obtaining a 1500 bp fragment for all strains which were cut by restriction enzymes selected by means of the analysis of sequences of the 16S ribosomal genes located in the Gene bank (bank of genes). With the RFLP-PCR a high specificity and sensibility were obtained since specific profiles for H. paragallinarum and different from those of O. rhinotracheale, Pasteurella spp. and E. coli were identified. The designed technique was used for the identification of 19 strains isolated from clinical samples of infraorbital sinus and nasal cornets of breeders and laying hens, and broiler chickens of diverse zones of Peru obtaining reproducible results and demonstrating the validity of this technique and its use for a rapid identification of microorganisms involved in respiratory infections in commercial birds. Key words: Haemophilus paragallinarum, Ornithobacterium rhinotracheale, Infectious Coryza, 16S ribosomal genes, RFLP-PCR. / Tesis

En el presente estudio se diseñó una técnica molecular alternativa para la identificación de Haemophilus paragallinarum, agente causal de la Coriza Infecciosa, basada en el polimorfismo en longitud de los fragmentos de restricción de los genes ribosómicos 16S amplificados por la reacción en cadena de la polimerasa (RFLP-PCR). Esta metodología fue propuesta debido a que las técnicas, HPG-1 y HPG-2, las cuales vienen siendo utilizadas en la identificación de este patógeno, mostraron baja sensibilidad y reacción cruzada con Escherichia coli, respectivamente. En la técnica HPG-2 se encontró dos productos de amplificación entre 450 y 500 pb confundiéndose con el producto de 500 pb que se obtiene con H. paragallinarum. Para la estandarización de la técnica molecular propuesta, se utilizaron las cepas de referencia ATCC 29545, B y C de H. paragallinarum, y como controles negativos a Ornithobacterium rhinotracheale, Pasteurella spp., microorganismos con características morfológicas, bioquímicas y cuadros clínicos similares a H. paragallinarum; y E. coli, esta última, asociada en la Coriza Infecciosa complicada. Se utilizaron cebadores universales para amplificar los genes ribosómicos 16S con los que se obtuvo un fragmento de 1500 pb para todas las cepas, éstos fueron cortados con enzimas de restricción seleccionadas mediante el análisis de las secuencias de los genes ribosómicos 16S depositadas en el GenBank (banco de genes). Con el RFLP-PCR se obtuvo una mayor especificidad y sensibilidad ya que se identificaron perfiles genéticos específicos para H. paragallinarum y diferentes a los de O. rhinotracheale, Pasteurella spp y E. coli. La técnica diseñada fue utilizada en la identificación de 19 cepas aisladas de muestras clínicas de senos infraorbitarios y cornetes nasales de gallinas reproductoras, de postura y pollos de carne de diversas zonas del Perú obteniendo resultados reproducibles y demostrando la validez de esta técnica y su uso para una rápida identificación de microorganismos implicados en infecciones respiratorias en aves comerciales. Palabras claves: Haemophilus paragallinarum, Ornithobacterium rhinotracheale, Coriza Infecciosa, genes ribosómicos 16S, RFLP-PCR. / In the present study, a technical molecular alternative for the identification of Haemophilus paragallinarum, causal agent of the Infectious Coryza, based on the restriction fragments length polymorphism of the 16S ribosomal genes amplified by the chain reaction polymerase (RFLP-PCR) was designed. This methodology was proposed due to the fact that the HPG-1 and HPG-2 techniques, which have been used for the identification of this pathogen, showed low sensitivity and crossed reaction with Escherichia coli respectively. In the HPG-2 technique, two amplification products between 450 and 500 bp were obtained, similar with the 500 bp product obtained with H. paragallinarum. For the standardization of the proposed molecular technique, we used the ATCC 29545 reference strain and the B and C strains of H. paragallinarum, as well as Ornithobacterium rhinotracheale, Pasteurella spp., microorganisms with similar morphological, biochemical characteristics and clinical signs to those of H. paragallinarum, and E. coli, bacterium associated in the complicated Infectious Coryza, as negative controls. Universal primers for amplifying the 16S ribosomal genes were used, obtaining a 1500 bp fragment for all strains which were cut by restriction enzymes selected by means of the analysis of sequences of the 16S ribosomal genes located in the Gene bank (bank of genes). With the RFLP-PCR a high specificity and sensibility were obtained since specific profiles for H. paragallinarum and different from those of O. rhinotracheale, Pasteurella spp. and E. coli were identified. The designed technique was used for the identification of 19 strains isolated from clinical samples of infraorbital sinus and nasal cornets of breeders and laying hens, and broiler chickens of diverse zones of Peru obtaining reproducible results and demonstrating the validity of this technique and its use for a rapid identification of microorganisms involved in respiratory infections in commercial birds. Key words: Haemophilus paragallinarum, Ornithobacterium rhinotracheale, Infectious Coryza, 16S ribosomal genes, RFLP-PCR.

Thesis Erasmus University Rotterdam. / ook verschenen in gedrukte versie. With bibliogr., with a summary in Dutch.