Tesi sul tema "Ticks microbiology"

The Lyme disease causing spirochaete Borrelia burgdorferi sensu lato is transmitted by ticks within the genus Ixodes. These ticks are liberal host seekers and parasitise mammals, birds and reptiles. Prior to this study, the distribution of I. ricinus ticks and Lyme disease was thought to be restricted to the southern half of Sweden. On the island Norrbyskär, located in the Bothnian Gulf, there were reports of a high incidence of tick infestation on humans. To investigate the occurrence of B. burgdorferi s.l. in these ticks and to characterise presumptive isolates at the molecular level we sampled a number of I. ricinus ticks. Three different isolates were obtained from two different ticks, NBS16 from a nymph and NBS23a and NBS23b from an adult female tick. The seabird associated tick I. uriae is circumpolar distributed in both hemispheres. On the island Bonden, which house one of the largest seabird colonies in the Baltic Sea, I. uriae were collected and surveyed for spirochaetes. One isolate of B. burgdorferi s.l. was obtained. This B. burgdorferi s.l. isolate is identical to the Lyme disease Borrelia strain NBS16 isolated from Norrbyskär. To investigate the role of seabirds in the epidemiology of B. burgdorferi s.l., I. uriae were collected from seabird colonies in the southern and northern hemispheres. Borrelia DNA was extracted from the ticks and from cultured spirochaetes. Sequence analysis of the flagellin gene revealed that the DNA obtained was from B. garinii, regardless of the geographical origin of the sample. Identical fla gene fragments in ticks collected in both hemispheres indicate a transhemispheric exchange of B. garinii. A marine ecological niche and epidemiological route for Lyme disease Borrelia are proposed. The prevalence of B. burgdorferi s.l. infected ticks on migrating passerine birds was studied. A total of 22, 998 birds were caught and examined for ticks. The presence of spirochaetes in the 967 collected ticks was determined by DNA amplification by PCR on all ticks. To determine which B. burgdorferi s.l. species were present, classification was performed by DNA amplification using species-specific 16S rDNA primers and by DNA sequencing. Flagellin gene sequences of all species of B. burgdorferi s.l. previously recorded in Europe were found. B. garinii was the most prevalent. These data support the notion that passerine birds are at least partly responsible for the distribution of Lyme disease Borrelia spirochaetes in Europe. To elucidate the distribution of B. burgdorferi s.l. in subarctic regions, strains isolated from I. ricinus and I. uriae ticks found on islands in the northern Atlantic and Baltic Sea were characterised molecularly. All isolates were verified as B. garinii by 16S-rRNA gene analysis and immunoblotting using monoclonal antibodies specific for the outer surface proteins A and C. Three ribotypes (RT's) of B. garinii were found. The I. ricinus associated RT1 is phenotypically the most heterogeneous. RT2 is restricted to the islands in the northern Baltic Sea, whereas RT3 was also recovered from ticks found on islands in the North Atlantic. The heterogeneity of the B. garinii population in the Baltic Sea might be influenced by two geographically opposite directions, North Atlantic (RT3) and Euroasia (RT1).
digitalisering@umu.se

This work presents an evaluation of the applicability of gene reporter technology to monitor Escherichia coli stress in industrial conditions with special interest in recombinant protein production. Different reporter plasmids containing promoter sequences of genes of the heatshock response were utilized to monitor chaperone expression upon different sources of stress such as exposure to chemicals, temperature and anaerobic growth. Activation of the heat shock response was monitored by \(\beta\)-galactosidase activity from the reporter plasmid pQF50groE. Cultures responded to heat-shock, anaerobiosis and \(\beta\)-mercaptoethanol by increasing the expression of \(\sigma\)\(^{32}\)-related genes. The performance of fluorescence reporters containing varieties of GFP was measured by fluorimetry and flow cytometry. Low copy number plasmids were demonstrated to be better suited than medium-high copy plasmids to report stress in industrial conditions. Reporter plasmids containing the promoters of the chaperones DnaK and GroES were utilized to measure E. coli stress in reducing environments and during recombinant protein production. It was demonstrated that the production strategy caused an impact in the host physiology which determined the outcome of the process. Flow cytometry showed excellent potential to obtain reliable measurements providing data of reporter activity cell death and cell morphology.

Flaviviruses are important emerging and re-emerging arthropod-borne pathogens that cause significant morbidity and mortality in humans. It consists of globally distributed human pathogens such as tick-borne encephalitis virus (TBEV), West Nile virus (WNV), Japanese encephalitis virus (JEV), yellow fever virus (YFV), dengue virus (DENV), and Zika virus (ZIKV). Depending on type, flaviviruses can cause a variety of symptoms ranging from haemorrhage to neurological disorders. Virus infection is detected by host pattern recognition receptors (PRRs), and through downstream signalling it leads to the production of interferons (IFNs). These IFNs then act in an autocrine or paracrine manner on the cells to induce various IFN-stimulated genes (ISGs), which have antiviral roles. However, the amount of IFN produced depends on the nature of the PRRs used by host cells to detect a particular virus. Although there are many PRRs present in the host cells, their relative contribution in different cell types and against a specific virus may vary. In the first study, we determined the importance of IPS-1 signalling in immunity and pathogenicity of tick-borne flaviviruses. This is an adaptor protein for cytoplasmic RIG-I-like receptors. Using IPS-1-deficient mice, we showed its importance against TBEV and Langat virus (LGTV) infection (the LGTV model virus belongs to the TBEV serogroup). Absence of IPS-1 leads to uncontrolled virus replication in the central nervous system (CNS), but it has only a minor role in shaping the humoral immune response at the periphery. LGTV-infected IPS-1-deficient mice showed apoptosis, activation of microglia and astrocytes, an elevated proinflammatory response, and recruitment of immune cells to the CNS. Interestingly, we also found that IFN-b upregulation after viral infection was dependent on IPS-1 in the olfactory bulb of the brain.  Thus, our results suggest that local immune microenvironment of distinct brain regions is critical for determination of virus permissiveness. Interferons can upregulate several ISGs. Viperin is one such ISG that has a broad-spectrum antiviral action against many viruses. However, the importance of cell type and the significance of viperin in controlling many flavivirus infections in vivo is not known. Using viperin-deficient mice, we found that viperin was necessary for restriction of LGTV replication in the olfactory bulb and cerebrum, but not in the cerebellum. This finding was also confirmed with primary neurons derived from these brain regions. Furthermore, we could also show the particular importance of viperin in cortical neurons against TBEV, WNV, and ZIKV infection. The results suggested that a single ISG can shape the susceptibility and immune response to a flavivirus in different regions of the brain. Although viperin is such an important ISG against flaviviruses, the exact molecular mechanism of action is not known. To understand the mechanism, we performed co-immunoprecipitation screening to identify TBEV proteins that could interact with viperin. While viperin interacted with the prM, E, NS2A, NS2B, and NS3 proteins of TBEV, its interaction with NS3 led to its degradation through the proteosomal pathway. Furthermore, viperin could reduce the stability of other viperin-binding TBEV proteins in an NS3-dependent manner. We screened for viperin activity regarding interaction with NS3 proteins of other flaviviruses. Viperin interacted with NS3 of JEV, ZIKV, and YFV, but selectively degraded NS3 proteins of TBEV and ZIKV, and this activity correlated with its antiviral activity against these viruses. The last study was based on in vivo characterization of the newly isolated MucAr HB 171/11 strain of TBEV which caused unusual gastrointestinal and constitutional symptoms. This strain was compared with another strain, Torö-2003, of the same European subtype of TBEV but isolated from the different focus. Here we found unique differences in their neuroinvasiveness and neurovirulence, and in the immune response to these two strains. In summary, my work shed some light on the interplay between tick-borne flavivirus and the innate immune system. I have shown two examples of CNS region-specific differences in innate immune response regarding both in IFN induction pathways and antiviral effectors. Furthermore, we have investigated the in vivo pathogenesis of a strain of TBEV that caused unusual gastrointestinal and constitutional symptoms.
Flavivirus finns spridda över hela världen och orsakar miljontals infektioner varje år. Några av de medicinsk mest viktiga flavivirusen är fästingburen encefalit virus (TBEV), West Nile virus (WNV), Japansk encefalit virus (JEV), gula febern (YFV) och Zika virus (ZIKV). Dessa virus kan orsaka olika komplikationer till exempel blödarfeber och hjärninflammation. Vid en infektion så upptäcker värdcellen virusinfektionen med hjälp av speciella receptorer, så kallade PRRs. Dessa finns i alla celler och känner igen viruskomponenter som normalt inte finns i en oinfekterad cell. När PRRs detekterar en virusinfektion svarar cellen med att tillverka ett signal protein interferon (IFN). IFN skickas ut ur cellen och hämmar virusinfektioner genom att sätta igång ett försvarsprogram i andra celler bestående av hundratals försvarsproteiner som kan motverka virusinfektionen. Vilka PRRs som behövs för att detektera ett virus är olika vid olika virusinfektioner. I första studien fann vi att IPS-1 är av yttersta vikt för skydda mot fästingburna flavivirus. IPS-1 är ett så kallat adapter protein som behövs för att två PRRs, RIG-I och MDA-5, ska kunna förmedla signaler som leder till IFN tillverkning. Med hjälp av möss som saknar IPS-1 fann vi att IPS-1 behövs för att tillverka IFN protein och skydda mot fästingburna flavivirus. IPS-1 var särskilt viktigt för interferon produktion inom luktloben i hjärnan. Därför kunde vi dra slutsatsen att immunresponsen regleras olika inom olika delar av hjärnan. Ett försvarsprotein som visat sig vara särskilt viktig vid virusinfektion är viperin. Viperin har visat sig kunna hämma en rad olika virus men den specifika rollen av viperin in vivo vid flavivirus infektion var inte fullt känd. Vi fann att viperin behövs för att hämma LGTV i lukloben och storhjärnan men inte i lillhjärnan. Vi kunde bekräfta detta med hjälp av primära nervceller isolerade från dessa hjärnregioner. Vi fann även att viperin var av yttersta vikt för att kontrollera TBEV, WNV och ZIKV infektion i nervceller från hjärnbarken (del av storhjärnan). Därför kunde vi dra slutsatsen att ett enskilt försvarsprotein kan avgöra mottagligheten mot flavivirus inom olika hjärnregioner. Trots att viperin är så viktig för att skydda mot flavivirus så vet vi inte hur viperin åstadkommer detta. Därför ville vi undersöka hur viperin kan förmedla sin antivirala effekt. Vi fann att viperin kan binda till flera TBEV proteiner, men att viperin specifikt kan bryta ner ett virusprotein som heter NS3. NS3 är väldigt viktigt för att flavivirus ska kunna etablera en infektion och kunna föröka sig. Eftersom vi visste att viperin kan hämma andra flavivirus ville vi veta om viperin även förstör NS3 från JEV, ZIKV och YFV. Vi upptäckte att viperin kunde binda till NS3 hos alla dessa flavivirus men att viperin specifikt förstörde TBEV och ZIKV NS3, intressant nog så kunde viperin endast hämma dessa virus infektioner men inte JEV och YFV. I den sista studien ville vi karaktärisera en ny TBEV stam som bara orsakar magoch tarmbesvär men inga neurologiska symptom. TBEV har aldrig tidigare visat sig kunna orsaka detta och därför ville vi undersöka saken vidare. Vi fann att denna TBEV stam skiljde sig mot en närbesläktad stam genom att orsaka en starkare immunrespons men mildare sjukdomsförlopp. Sammanfattningsvis har jag undersökt samspelet mellan fästingburna flavivirus och det medfödda immunförsvaret. Jag har även visat att immunresponsen regleras olika inom olika hjärnregioner, både beträffande IFN inducering och antivirala proteiner. Vidare har jag hittat mekanismen för hur viperin proteinet hämmar TBEV och ZIKV, vilket var genom att förstöra NS3. Dessutom har jag karaktäriserat sjukdomsförloppet hos möss efter infektion med en ovanlig TBEV stam som orsakar mag och tarm besvär.

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The National School Nutrition Program is the oldest program in the country directed at food and nutrition safety. Its aims are to offer nutritional food as a supplement to students of public and philanthropic schools. Studying school nutrition transcends the investigation as a purely social program, given that it reaches the fields of public health, nutrition and food, using a wide variety of approaches. Thus, it is characterized by a multidisciplinary study, where the disciplines work side by side in distinct aspects of a single problem. Aim: This study aims to assess hygiene practices during the preparation of meat-based meals in public schools in the city of Natal, Brazil. Methods: A list was applied at 27 schools to identify the procedures of good food preparation practices. In addition, cooking and meal distribution temperature were measured and a microbiological analysis of the final preparation and of the water used in preparing it was performed. For microbiological analyses of the food, we analyzed coliforms at 45?C, coliforms at 35?C and Enterococcus, and for the water, we analyzed thermotolerant coliforms and total coliforms, using the methods recommended by APHA, 1995. Results: Most of the schools did not meet the required standards in all the variables related to good food preparation practices, except for the time spent preparing the meat, in which 89% were within the norm. Cooking temperature of the meals was within the standard; however, the temperature at distribution and the time spent dispensing the meals were inadequate. Of the 27 schools, 22 (81.5%) showed the presence of coliforms at 35? C in at least one meal sample and 18 (66.7%) had values above the recommended limit for coliforms at 45?C. The presence of E. coli was identified in 6.1% of the samples analyzed. The presence of Enterococcus was not found at any of the schools. With respect to the water, the North district of the city was the only one that did not meet the standards for the two indicators evaluated. The contamination found was not associated with the hygiene or food storage problems observed. Conclusions: The results show that the hygiene-sanitary conditions of meat-based public school meals were unsatisfactory, demonstrating the need for improvements in the production process to preserve the health of the student population. Multidisciplinarity: Researchers from the areas of food microbiology, nutrition, public health and statistics took part in this study, a decisive factor for characterizing the research as multidisciplinary
O Programa Nacional de Alimenta??o Escolar ? o programa voltado ? seguran?a alimentar e nutricional mais antigo do pa?s e tem por objetivo oferecer alimentos, de qualidade, em car?ter suplementar aos estudantes de escolas p?blicas e filantr?picas. Estudar a alimenta??o escolar transcende a investiga??o enquanto programa social, atingindo os campos da sa?de p?blica, nutri??o e alimentos, nas suas mais variadas abordagens. Assim, caracteriza-se por um estudo de car?ter multidisciplinar, onde as disciplinas trabalham lado a lado em distintos aspectos de um ?nico problema. Objetivo: O presente trabalho tem como objetivo avaliar as pr?ticas de higiene durante a produ??o de prepara??es ? base de carne em escolas p?blicas municipais na cidade de Natal/RN. M?todos: Foram avaliadas 27 escolas, onde foi aplicada uma lista de verifica??o a fim de identificar os procedimentos de Boas Pr?ticas de Fabrica??o, e ainda, foi medida a temperatura de coc??o e distribui??o das prepara??es e realizada an?lise microbiol?gica da prepara??o pronta e da ?gua utilizada no preparo das mesmas. Para as an?lises microbiol?gicas do alimento, foram analisados coliformes ? 45?C, coliformes ? 35?C e Enterococcus, e para a ?gua, foram analisados coliformes termotolerantes e coliformes totais, atrav?s dos m?todos preconizados pela APHA, 1995. Resultados: A maioria das escolas estudadas apresentou n?o conformidades em todas as vari?veis analisadas quanto ?s Boas Pr?ticas de Fabrica??o, com exce??o do tempo de pr?-preparo das carnes, no qual 89% estavam dentro do padr?o. A temperatura de coc??o das prepara??es encontrou-se dentro do padr?o, entretanto a temperatura e o tempo de distribui??o apresentaram-se inadequados. Das 27 escolas, 22 (81,5%) apresentaram pelo menos uma amostra da prepara??o pronta com presen?a de coliformes ? 35?C, e 18 (66,7%) apresentaram valores acima do padr?o para coliformes ? 45?C. Foi identificada a presen?a de E. coli em 6,1% das amostras analisadas. N?o foi encontrada a presen?a de Enterococcus em nenhuma escola. Com rela??o ? ?gua, a regi?o Norte foi a ?nica com evid?ncias estat?sticas de estar fora do padr?o para os dois indicadores avaliados. A contamina??o encontrada n?o apresentou associa??o com as n?o conformidades referentes ? higiene peri?dica e a conserva??o dos reservat?rios. Conclus?es: Os resultados encontrados mostram que as condi??es higi?nico-sanit?rias das prepara??es ? base de carne servidas na alimenta??o escolar apresentaram-se insatisfat?rias, evidenciando a necessidade de melhoria do processo de produ??o, visando ?s condi??es de sa?de da popula??o estudantil assistida. Multidisciplinaridade: Este estudo teve a participa??o de pesquisadores das ?reas de microbiologia de alimentos, nutri??o, sa?de p?blica e estat?stica, fator decisivo para caracterizar a pesquisa como multidisciplinar

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Streptococcus agalactiae, also referred as group B streptococci (GBS) are commensals microorganisms adapted to asymptomatic colonization of the mammalians gut and genitourinary tract. Initially, this specie was recognized as a major etiologic agent for bovine mastitis but has becoming a leading cause of invasive infections in human neonates. The reasons behind the prompt and persistent emergence of GBS neonatal disease have not been completely elucidated, once human and bovine GBS populations are assumed be distinct and unrelated by divergence on their own physiological characters. Aiming to contribute for the characterization of the two S. agalactiae sub-populations that are in a proximal coexistence in the Rio de Janeiro state, this study evaluated phenotypic and genotypic diversity aspects of regional groups of GBS. The first made up of 50 isolates obtained from human specimens whilst the other group was constituted of 36 isolates from milk of dairy cows presenting clinical or sub clinical mastitis. Phenotypic characterization was based on physiological and serological tests, antimicrobial susceptibility assays were carried out by the disk standard procedure and microdilution method. The genetic aspect was assessed by PCR for detection of genes associated with resistance to tetracycline. According to the results of physiologic tests, β-hemolysis was a faculty shared by about 28% of bovine isolates and 100% of human isolates. GBS bovine isolates also shows different profile of sensitivity to bacitracin, only 33% of them were susceptive to the antibiotic, regardless of the whole human isolates set had demonstrated a 100% susceptive pattern to this substance. A 100% sensitivity percentual to penicillin was shared by all isolates assayed in this study corroborating the general procedure for antibiotic therapy of GBS infection. Otherwise, and in an overall view, bovine isolates showed higher resistance rates to a set of antibiotics, including cephoxitin, erytromicin, clyndamicin, sulphamethoxazole, azithromycin and ciprofloxacin than in their human counterparts. In a similar sense, it was observed in this study that 13,9% of the animal GBS isolates expressed cMLSB and 2,8 % M phenotypes. The M phenotype was expressed in 6% of the human related isolates as the unique MLSB parameter. Genetical assays performed detected 13,8% (5/36) and 14% (7/50) for tet (M), and 30,5% (11/36) and 10% (5/50) for de tet (O), respectively, in bovine and human isolates. These genes are implicated in tetracycline resistance by ribosome protection mechanism through enzymatic structural modification.
Os Streptococcus agalactiae, tamb?m designados como estreptococos do grupo B (EGB), s?o microrganismos comensais adaptados para fazer a coloniza??o assintom?tica do tubo digestivo, e do trato geniturin?rio de mam?feros. Inicialmente, reconhecida como um dos mais importantes agentes etiol?gicos da mastite bovina, esta esp?cie foi tamb?m implicada como uma das principais causas de infec??es invasivas em rec?m-nascidos humanos. As raz?es para a r?pida e consistente evolu??o do EGB como importante agente causal de infec??es neonatais ainda n?o foram completamente elucidadas, uma vez que as subpopula??es de EGB nos isolados de humanos e bovinos s?o independentes e distintas com base no reconhecimento da diversidade de suas caracter?sticas fisiol?gicas. Com o objetivo de contribuir para a caracteriza??o das duas subpopula??es de S. agalactiae que coexistem no Estado do Rio de Janeiro, este estudo avaliou aspectos da diversidade gen?tica e fenot?pica de dois grupos de EGB regionais, sendo o primeiro composto por 50 isolados obtidos a partir de esp?cimes cl?nicos humanos, e o segundo constitu?do por 36 isolados a partir de leite de vacas leiteiras com ind?cios de mastite cl?nica ou subcl?nica. A caracteriza??o fenot?pica dos isolados foi baseada em testes sorol?gicos e fisiol?gicos, testes de suscetibilidade antimicrobiana realizados com t?cnica padronizada para utiliza??o de disco de difus?o e pelo m?todo de microdilui??o. O aspecto gen?tico foi avaliado pela aplica??o de PCR para detec??o de genes associados ? resist?ncia ? tetraciclina. Os testes fisiol?gicos demonstraram que a capacidade de promover β-hem?lise era uma caracter?stica partilhada por cerca de 28% dos isolados a partir do material de natureza bovina, mas que manifestava-se em todos os isolados de origem humana. Os isolados de EGB bovinos tamb?m mostraram um perfil diferente quanto ? sensibilidade ? bacitracina, uma vez que apenas 33% delas se revelaram suscet?veis a esse antibi?tico contra 100% de sensibilidade para os isolados de origem humana. O percentual de 100% de sensibilidade ? penicilina demonstrado por todos os isolados analisados neste estudo, tamb?m corrobora a import?ncia do uso desse antibi?tico como procedimento geral na terapia de infec??es por EGB. De uma forma geral, neste estudo foi observado que os isolados originados de material bovino demonstraram percentuais de resist?ncia ao conjunto de antibi?ticos analisados (cefoxitina, eritromicina, clindamicina, sulfametoxazol, azitromicina e ciprofloxacina) superiores aos observados em isolados de material humano. Foi tamb?m observado que 13,9% dos isolados de EGB animais examinados expressaram o fen?tipo cMLSB e 2,8% o fen?tipo M. O fen?tipo M foi o ?nico par?metro MLSB expresso entre os isolados de S. agalactiae humanos, com um percentual de 6%. Quanto ? presen?a de genes de resist?ncia a tetraciclina entre as subpopula??es de EGB, detectou-se percentuais de 13,8% (5/36) e 14% (7/50) para tet(M), e 30,5% (11/36) e 10% (5/50) para tet(O), respectivamente, nos isolados bovinos e humanos avaliados. Estes genes est?o implicados na resist?ncia ? tetraciclina por um mecanismo prote??o ribossomal, pela altera??o estrutural mediada por a??o enzim?tica. Palavras-chave: Streptococcus agalactiae, caracteriza??o fenot?pica, gene tet

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Funda??o de Amparo ? Pesquisa do Estado da Bahia - FAPEB
Pineapple is a tropical fruit of great commercial value, Brazil is the second largest producer of pineapple in the world. The most produced variety in the country is the ?Perola?, followed by Smooth Cayenne and, both have a high degree of sus-ceptibility to fusariosis or pineapple gum disease, caused by the fungus Fusarium guttiforme.Once installed, this disease can reduce pineapple yield by up to 80%. Lactic bacteria (BAL) have their metabolic characteristics and the potential to inhibit bacterial and fungal growth, as well as to neutralize toxins produced by F. guttiforme.This work aimed to determine by in vitro tests the inhibitory effect of lactic acid bacteria on the etiologic agent of fusariosis. Qualitative and quantitative microbial inhibition tests were performed, comparing strains of lactic acid bacteria and crude pineapple extract against F. guttiforme isolates. Determination of mycotoxins in the fungal extracts of these isolates. The inhibitory effect of Lactobacillus plantarum Abx3 and Lactobacillus paracasei subsp.paracasei Abx5.2 was observed for isolateson F. guttiforme, the same did not occur with crude pineapple extract, and mycotoxin production was not detected in extracts of fungal isolates. The preliminary results of this work point out that lactic bacteria present potential to be used as a biocontrol product on F. guttiforme.
O abacaxi ? uma fruta tropical de grande valor comercial, sendo o Brasil o se-gundo maior produtor no mundo. A variedade mais produzida no pa?s ? a P?rola, seguida pela Smooth Cayenne, ambas possuem alto grau de suscetibilidade ? fusa-riose ou gomose do abacaxi, causada pelo fungo Fusarium guttiforme. Esta doen?a, uma vez instalada, pode reduzir em at? 80% a produ??o dos cultivares. As bact?rias l?ticas (BAL), devido as suas caracter?sticas metab?licas possuem potencial de inibir o crescimento bacteriano e f?ngico, bem como neutralizar toxinas produzidas por algunsmicro-organismos. Este trabalho teve como objetivodeterminar atrav?s de tes-tes ?in vitro? o efeito inibit?rio de bact?rias l?cticas sobre o agente etiol?gico da fusa-riose do abacaxi.Foram realizados ensaios qualitativo e quantitativode inibi??o mi-crobiana, confrontando cepas de bact?rias l?ticas e extrato bruto do abacaxifrente aisolados de F.guttiforme. Determinarse h? micotoxina nos extratos f?ngicos destes isolados. Constatou-se efeito inibit?rio do Lactobacillus plantarum Abx3 e Lactobacillus paracaseisubsp.paracasei Abx5.2 sobre os isolados de F. guttiforme. O mesmo n?o aconteceu com o extrato bruto de abacaxi, e n?o foi detectada a produ??o de micotoxina nos extratos dos isolados f?ngicos. Os resultados preliminares deste trabalho apontamque as bact?rias l?ticas apresentam potencial de serem utilizadas em formula??es de produto de biocontrolede F. guttiforme.

Dans ce travail, nous nous sommes intéressés à l’étude des infections à Anaplasmataceae chez les animaux et leurs tiques afin de caractériser au mieux ces infections animales et humaines et décrire de nouvelles espèces. Nous avons tout d’abord proposé un système de diagnostic moléculaire qui couple une qPCR suivie d’amplification et un séquençage ciblant le gène 23S ARNr. Puis, au long de ce travail, nous avons proposé d’autres amorces ciblant d’autres gènes incluant la sous unité ribosomal bêta (rpoB), la protéine du choc thermique (groEl), et le 16S ARNr. Notre objectif a été de sélectionner et d’identifier les différentes espèces impliquées ou non dans des pathologies chez les animaux et mettre en évidence leur vecteur. Au cours de ces travaux, nous avons eu accès à différents prélèvements de sang et des tiques en provenance de diverses régions du monde incluant la France métropolitaine et d’outre-mer, l’Algérie, le Niger, la Côte d’Ivoire, le Sénégal et le Pakistan. Nos investigations ont permis d’identifier différentes espèces d’Anaplasmataceae incluant de potentielles nouvelles espèces. Les prévalences rapportées dans chaque étude démontrent que les animaux sont les réservoirs de ces infections. Les recherches menées sur les tiques ont permis d’identifier de potentiels vecteurs d’Anaplasmataceae dans différentes régions du monde. Les potentielles nouvelles espèces identifiées sont caractérisées en ciblant différents gènes, et les analyses moléculaires démontrent qu’elles sont différentes des autres Anaplasmataceae connues jusqu’à maintenant. Ces différents travaux apportent donc davantage d’informations sur l’épidémiologie des Anaplasmataceae dans le monde
In this work, we are interested in studying Anaplasmataceae infections in animals and their ticks. Our objective is to describe these infections in animals and to identify new species implicated in different pathology. First, we propose a molecular diagnostic approach that couples a qPCR followed by amplification and sequencing targeting the 23S rRNA gene. Then we propose other primers targeting other genes including the ribosomal subunit beta (rpoB), heat shock protein (groEl), and the 16S rRNA. Our goal was to screen and identify the different species involved, or not involved, in pathologies of animals and identify their vectors. During this work, we had access to different blood samples and ticks from different parts of the world including metropolitan France, France overseas, Algeria, the Republic of Niger, Côte d'Ivoire, Senegal and Pakistan. Our different investigations allowed to identify different species of Anaplasmataceae including potential new species. The prevalence reported in each study demonstrates that animals are the reservoirs of these infections. So, the research conducted on ticks has identified potential vectors of Anaplasmataceae in different regions of the world. Potentially new species were identified are characterized by different targeting genes. These studies provide further information on the epidemiology of Anaplasmataceae in the world

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Conselho Nacional de Pesquisa e Desenvolvimento Cient?fico e Tecnol?gico - CNPq
Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES
This project applied the DNA metabarcoding technique, which consists of a large-scale identification of the biodiversity present in a given community (through the total DNA sequencing without the need for isolation and laboratory cultivation) in two underexplored Brazilian environments with high biological diversity. The first is a typical Neotropical microenvironment, called bromeliad phytotelm, which is formed by the accumulation of water and debris among the leaves of these plants. The bromeliads investigated in this study were sampled in areas of dense ombrophilus forest and mixed ombrophilus forest in southern Brazil (located at the ?Pro-Mata? Research Center), and belonged to the species Aechmea gamosepala Wittmack, Vriesea friburgensis Mez and Vriesea platynema Gaud. The collection comprised multiple individuals per species and also multiple samples (different tanks) from the same individual, making it possible to assess the complexity of the variation in these communities. The second environment consists of marine sediments collected in the Rio Grande Cone (located in southeastern Brazil, in the offshore portion of the Pelotas Basin), at depths of down to 18 meters below the seafloor. The samples originated from four different areas, with different geochemical characteristics and strong indications of the presence of chemosynthetic communities. This study demonstrated the presence of high biodiversity in both environments. We identified 30 prokaryotic phyla and 67 eukaryotic phyla in bromeliad phytotelmata, which seem to include both endemic organisms of this peculiar environment and organisms with ubiquitous distribution, common in freshwater or soil habitats. An interesting feature is that, in some cases, samples from different tanks of the same individual are more similar to tanks of other individuals (and even from other species) than between them. Thus, our results suggest that each bromeliad tank acts as an isolated body of water, where the effects of predation, competition and stochastic events such as wind-borne particles, fecal pellets and liquid excretions of terrestrial animals, dead leaves and animals can largely drive the community composition. Concerning the marine sediments, we observed the presence of 58 prokaryotic phyla, many of which are present in other chemosynthetic communities. Among these organisms were identified anaerobic methanotrophic archaeal groups and their syntrophic partners (sulfate-reducing bacteria), which together form a consortium with a significant role in the anaerobic oxidation of methane, a very important process that controls the emission of this greenhouse gas into the atmosphere. Statistical analysis performed in both studies sought to describe patterns of diversity of these communities at different spatial scales and to interpret them in the light of current knowledge about these environments. Overall, the results obtained in this thesis reveal in detail the complexity of these communities, opening new ways for further studies focusing on the processes that control their spatio-temporal dynamics.
O presente projeto aplicou a t?cnica de DNA metabarcoding, que visa caracterizar em larga escala a biodiversidade presente em determinada comunidade (atrav?s do sequenciamento do DNA total e sem a necessidade de cultivo ou isolamento), em dois ambientes brasileiros pouco explorados e com grande riqueza de esp?cies. O primeiro ? um microambiente t?pico da regi?o neotropical, denominado fitotelmo de brom?lia, o qual ? formado pelo ac?mulo de ?gua e detritos entre as folhas destas plantas. As brom?lias investigadas neste estudo foram amostradas em ?reas de Floresta Ombr?fila Densa e/ou Mista no sul do Brasil (localizadas no Centro de Pesquisas e Conserva??o da Natureza Pr?-Mata, PUCRS), e pertencem ?s esp?cies Aechmea gamosepala Wittmack, Vriesea friburgensis Mez e Vriesea platynema Gaud. A coleta englobou m?ltiplos indiv?duos por esp?cie e tamb?m m?ltiplas amostras (diferentes cisternas) do mesmo indiv?duo, possibilitando avaliar a complexidade da varia??o nestas comunidades. O segundo ambiente consiste de sedimentos marinhos coletados no Cone de Rio Grande (situado no sudeste do Brasil, na por??o offshore da Bacia de Pelotas), em profundidades de at? 18 metros abaixo do fundo do mar. As amostras s?o provenientes de quatro ?reas distintas, apresentando diferentes caracter?sticas geoqu?micas e fortes ind?cios da presen?a de comunidades quimiossint?ticas. Nossa investiga??o observou grande diversidade biol?gica em ambos os ambientes. Foram identificados 30 filos de procariotos e 67 filos de eucariotos nos fitotelmos de brom?lia, os quais parecem compreender tanto organismos end?micos deste ambiente peculiar como organismos com distribui??o cosmopolita, comuns em habitats de ?gua doce ou solo. Uma caracter?stica interessante ? que, em alguns casos, amostras provenientes de diferentes cisternas do mesmo indiv?duo s?o mais semelhantes a cisternas de outros indiv?duos (e mesmo de outras esp?cies) do que entre si. Desta forma, nossos resultados indicam que cada cisterna atua como um corpo d??gua isolado, onde os efeitos de preda??o, competi??o e eventos estoc?sticos como part?culas carregadas pelo vento, restos de folhas ou animais mortos, bem como excre??es de animais, podem interferir fortemente na composi??o espec?fica da comunidade local. Em rela??o aos sedimentos marinhos, observamos a presen?a de 58 filos procari?ticos, os quais compreendem diversos t?xons j? caracterizados em outras comunidades quimiossint?ticas marinhas. Entre estes organismos, foram identificadas arqueas metanotr?ficas e seus parceiros sintr?ficos (bact?rias redutoras de sulfato), que juntos formam um cons?rcio cujo papel ? fundamental na oxida??o anaer?bica do metano, um importante processo que controla a emiss?o deste g?s de efeito estufa para a atmosfera. An?lises estat?sticas realizadas em ambos os estudos buscaram descrever padr?es de diversidade destas comunidades em diferentes escalas espaciais, bem como interpret?-los ? luz do conhecimento atual sobre estes ambientes. De forma geral, os resultados obtidos nesta tese revelam em detalhe a complexidade destas comunidades, e abrem caminho para estudos mais aprofundados dos processos que controlam sua din?mica espa?o-temporal.

Ce travail s’articule sur trois axes ; le premier est une contribution à l'étude du répertoire des bactéries associées aux arthropodes vecteurs (tique et puces) en Afrique du nord (Algérie) et en Afrique Sub-saharienne (Bénin, Tanzanie et République Démocratique du Congo). Nous avons pu ainsi détecter par biologie moléculaire (qPCRs, PCR standard et séquençage) et pour la première fois au Bénin, Rickettsia typhi (l'agent du typhus murin), et Bartonella sp dans des puces collectées sur des rongeurs à Cotonou. Dans ce travail, nous avons également détecté Yersinia pestis, l'agent de la peste et R. felis (responsable de la fièvre boutonneuse) dans des puces de la RD du Congo. En Tanzanie, nous avons mis en évidence la présence de R. felis et R. typhi dans des puces de rongeurs. En Algérie, nous avons décrit pour la première fois la présence d'agent de borréliose de Lyme (Borrelia garinii) dans les tiques. Nous avons confirmé la présence de R. massiliae, R. monacensis R. aeschlimannii, R. slovaca et R. felis et nous avons également détecté pour la première fois en Algérie, Bartonella tamiae, une bactérie dont la pathogénicité est peu connue et Coxiella burnetii, l'agent de la fièvre Q.Dans la deuxième partie de notre travail, nous nous sommes intéressés à l’évaluation des compétences vectorielles des puces de chat (Ctenocephalides felis) et punaises de lit (Cimex lectularius) dans la transmission de l’agent de la fièvre des tranchées (Bartonella quintana) dont le vecteur connu est le pou de corps. Trois approches ont été utilisées : la qPCR, la culture et l’immunohistochimie
This work focuses on three areas; the first is a contribution to the study of the repertoire of bacteria associated with arthropod vectors (tick and flea) in North Africa (Algeria) and in Sub-Saharan Africa (Benin, Tanzania and the Democratic Republic of Congo). We could thus detected by molecular tools (qPCRs, standard PCR and sequencing) and for the first time in Benin, Rickettsia typhi (the agent of murine typhus) and Bartonella sp in fleas collected from rodents in Cotonou. In this work, we have also associated the agent of plague (Yersinia pestis), and for the first time in fleas of DR of Congo, and we detected also R. felis (the causative agent of spotted fever). In Tanzania, we have highlighted the presence of R. typhi and R. felis fleas on rodents. In Algeria, we described for the first time the presence of Lyme disease agent (Borrelia garinii) in hard ticks. We confirmed the presence of R. massiliae, R. monacensis, R. aeschlimannii, R. slovaca and R. felis, we also detected for the first time Bartonella tamiae and Coxiella burnetii associated with bat ticks in Algeria.Regarding the second part we was interested in the evaluation of vector competence of cat fleas (Ctenocephalides felis) and bed bugs (Cimex lectularius) in the transmission of trench fever agent (Bartonella quintana) that is known to be transmitted by lice. Three approaches have been tested; qPCR, culture and immunohistochemistry

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Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior, CAPES, Brasil.
The increasing demand for biological processes alternative, environmentally friendly and efficient in converting lignocellulosic material, expanding their application potential for agribusiness, motivates researches worldwide. Thus, organisms isolated in nature, in specific ecosystems, become increasingly important because of their physiological and metabolic diversity, which gives them a great potential in the development of biotechnological processes of interest to society. The aim of this study was to assess the microbial community associated with the intestinal tract of millipede Trigoniulus corallinus and bioprospecting for microorganisms with cellulolytic capacity. The millipedes were collected and incubated with litter in diets of grass (Paspalum notatum) and ?sabia? (Mimosa caesalpinifolia). Sampling occurred at 15, 30, 45, and 75 days of incubation. The intestinal tract of five individuals was removed, sectioned the posterior third, processed and stored in ultrasound. DNA from microbes associated with the intestinal tract, litter and coprolite was extracted, and DGGE analysis using 16S rDNA, DGGE group actinomycetes, and it was evaluated the presence of nifH genes. The 16s gene analysis by DGGE revealed a microbial diversity conditioned by the diet offered to 45 days. After this period, this effect was no longer visible. The community associated with coprolites and the type of litter was distributed in separate clusters of samples from the intestinal tract. This effect was not observed in the community assessment of actinomycetes, where the big difference for division of groups was the diet. The animals fed on grass litter showed a diverse community, and they were not influenced by time or compartmentalization. The samples associated with litter and coprolites were 80% similar to samples from the intestinal tract. In millipedes fed with material form Mimosa caesalpinifolia, the result was different, the samples of litter and coprolites where 50% similar to the intestinal tract. All samples had nifH genes detected by polymerase chain reaction. Samples collected at 45 days were also inoculated in mineral minimum medium of Busnell-Hass added carboxymethyl-cellulose (CMC) as sole carbon source. Colonies were evaluated for their ability to breakdown cellulose enzyme and 15 had an index greater than 1. The isolate that showed the highest rate (3.65) was subjected to further analysis. The microscope observation suggested that this was not an isolated but a complex of microorganisms acting on the degradation of cellulose. There is evidence of BNF in the intestinal tract of the millipede and microorganisms proliferated in CMC through the proper amplification of nifH genes and proliferation in medium within nitrogen. The community of prokaryotes was influenced by the diet offered to the community up to 45 days, and the actinomycetes community was conditioned by the diet. It was possible to isolate microorganisms and complexes of microorganisms with cellulolytic capacity, with great potential in the search for environmentally friendly technologies in generating agrobioenergy.
A crescente demanda por processos biol?gicos alternativos, ambientalmente favor?veis e eficientes na transforma??o de material ligninocelul?sico, ampliando seu potencial de aplica??o agroindustrial, estimula pesquisas em todo o mundo. Assim, microrganismos isolados na natureza, em ecossistemas espec?ficos, tornam-se cada vez mais importantes pela sua diversidade metab?lica e fisiol?gica, que lhes confere grande potencialidade no desenvolvimento de processos biotecnol?gicos de interesse ? sociedade. O objetivo deste trabalho foi avaliar a comunidade microbiana associada ao trato intestinal do dipl?pode Trigoniulus corallinus e a bioprospec??o de microrganismos com capacidade celulol?tica. Os dipl?podes foram coletados e incubados em dietas com serrapilheira de grama batatais (Paspalum notatum) e sabi? (Mimosa caesalpinifolia). As amostragens aconteceram aos 15, 30, 45 e 75 dias de incuba??o. O trato intestinal de cinco indiv?duos foi removido e seccionado o ter?o posterior tratado em ultrasom e estocado. Procedeu-se a extra??o de DNA da microbiota associada ao trato intestinal, serrapilheira e copr?lito, com an?lise por DGGE utilizando o gene 16S rDNA, DGGE para grupo actinomicetos e avalia??o da presen?a de genes nifH. A an?lise do gene 16s por DGGE revelou diversidade microbiana condicionada pela dieta oferecida at? os 45 dias. Ap?s este per?odo o efeito n?o foi mais vis?vel. A comunidade associada aos copr?litos e ao tipo de serrapilheira distribui-se em grupamentos separados das amostras oriundas do trato intestinal. O mesmo n?o foi observado na avalia??o da comunidade de actinomicetos, onde o grande diferencial para divis?o de grupos foi a dieta. Os animais alimentados com serrapilheira de grama mostraram uma comunidade diversa e n?o influenciada pelo tempo ou compartimentaliza??o. As amostras associadas ? serrapilheira e aos copr?litos foram 80% similares ?s do trato intestinal. Nos dipl?podes alimentados com sabi?, o resultado foi diferente, sendo as amostras de serrapilheira e copr?litos 50% similares ?s do trato intestinal. Todas as amostragens tiveram genes nifH detectados via PCR. Amostras coletadas aos 45 dias foram tamb?m inoculadas em meio mineral m?nimo de Busnell-Hass adicionado de carboxi-metil-celulose (CMC) como ?nica fonte de carbono. Os microrganismos isolados foram avaliados quanto ? capacidade de degrada??o de celulose e 15 apresentaram ?ndice enzim?tico maior que 1. O isolado com o maior ?ndice (3,65) foi alvo de outras an?lises. A visualiza??o em microsc?pio sugeriu que n?o se tratava de um isolado e sim de um complexo de microrganismos atuando na degrada??o da celulose. H? evidencias de FBN no trato intestinal do dipl?pode e microrganismos proliferados em meio CMC pela boa amplifica??o de genes nifH e prolifera??o em meio com aus?ncia de nitrog?nio. A comunidade de procariotos foi influenciada pela dieta oferecida at? os 45 dias e a comunidade de actinomicetos foi condicionada em fun??o da dieta. Foram isolados microrganismos e complexos de microrganismos com capacidade celulol?tica, com grande potencial para a busca de tecnologias ambientalmente sustent?veis na gera??o de agrobioenergia.

Les maladies fébriles y compris les maladies bactériennes sont mal connues en Côte d’Ivoire et en Guinée. Tout d’abord, nous avons recherché par biologie moléculaire des bactéries pathogènes transmises par les tiques en Côte d’Ivoire. Nous avons analysé différentes espèces de tiques prélevées chez des bovins et mis en évidence des bactéries pathogènes responsables de nombreuses maladies infectieuses comme Rickettsia, Borrelia, Anaplasma, Ehrlichia, Coxiella burnetii (fièvre Q) et aussi vingt nouvelles espèces potentielles.Ensuite, notre objectif était de détecter par biologie moléculaire des micro-organismes pathogènes chez l’homme. Concernant l’étude des plaies et des peaux saines en Guinée, la plupart des patients étaient infectés par Pseudomonas aeruginosa, Staphylococcus aureus et plusieurs espèces d'Acinetobacter.Parmi les patients fébriles et les sujets apyrétiques recrutés en Guinée et en Côte d’Ivoire, Plasmodium falciparum reste le micro-organisme le plus fréquent surtout dans les échantillons de sang des patients fébriles bien que plusieurs bactéries aient été aussi identifiées. En Guinée, il s’agissait de Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Salmonella enterica Non Typhi et Non Paratyphi et R. felis. Ces bactéries ont été également identifiées ainsi que Salmonella enterica Typhi, Salmonella enterica Paratyphi, Tropheryma whipplei et une nouvelle espèce potentielle de Wolbachia en Côte d’Ivoire. Nos travaux ont permis d’établir le répertoire des bactéries transmises par les tiques en Côte d’Ivoire, celles impliquées dans les bactériémies en Côte d’Ivoire et en Guinée (Conakry)
Febrile illnesses including bacterial diseases are poorly known in Côte d'Ivoire and Guinea.In the first part of our work, we researched by molecular biology bacteria transmitted by ticks in Côte d’Ivoire. We analyzed different species of ticks collected from cattle and highlighted pathogenic bacteria responsible for many infectious diseases such as Rickettsia, Borrelia, Anaplasma, Ehrlichia, Coxiella burnetii (Q fever) and twenty potential new species. In the second part, our goal was to detect using molecular biology several microorganisms in humans in Guinea (Conakry) and Côte d'Ivoire. As regards the study of wounds and healthy skin in Guinea, most patients were infected with Pseudomonas aeruginosa, Staphylococcus aureus, several species of Acinetobacter.Among the febrile patients and healthy controls afebrile recruited in Guinea and Côte d'Ivoire, Plasmodium falciparum is the most common detected microorganism especially in blood samples from febrile patients although several bacteria were also identified. In Guinea, it was Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, non-typhoidal Salmonella spp., and R. felis. These bacteria were also identified as well as Salmonella enterica Typhi, Salmonella enterica Paratyphi, Tropheryma whipplei and a potential new species of Wolbachia in Côte d’Ivoire.This work allowed establishing the repertory of bacteria transmitted by ticks in Côte d’Ivoire, as well as those involved in bacteremia in Côte d’Ivoire and Guinea (Conakry)

Invertebrates do not possess an adaptive immune system, but rely on several mechanisms similar to the innate immune system of mammals. The synthesis and release of a host of potent antimicrobial proteins is an important component of this immune response. The antibacterial activity in the hemolymph of Ornithodoros savignyi is specific for Gram-positive bacteria, and the synthesis and release of the antibacterial factors need to be induced by challenging the ticks with heat-killed Gram-negative bacterial suspensions. The induction of the factors is very rapid, leading to a maximal response within one hour following bacterial challenge. The factors are stable at high temperatures, and were found to be protein in nature. By using reverse phase high performance liquid chromatography, four fractions exhibiting antibacterial activity were identified in the hemolymph of immune challenged ticks. Four antibacterial peptides were isolated from these fractions, and the mass analyses of the peptides indicate that there are at least two different antibacterial peptides present in the hemolymph. The N-terminal amino acid sequence of one of the peptides was determined, and the analysis showed that the peptide has high homology with defensin peptides isolated from other tick species. This led to the putative classification of the peptides as part of the invertebrate defensin family. The presence of lysozyme in O. savignyi was studied using molecular biological methods. Vertebrate and invertebrate lysozyme sequences were used to design a lysozyme-specific primer, which was used to amplify specific DNA products from whole tick cDNA using the polymerase chain reaction (PCR). The conditions for the amplification reaction were optimized, the products of the optimized reaction were cloned into a cloning vector and the nucleotide sequences of the products were determined. The nucleotide sequences were used for similarity searches of sequence databases to determine homology with sequences of known proteins. It is deduced the degenerate primer was not specific for lysozyme and did not playa significant role in the amplification of the PCR products. This method is thus not feasible for the investigation of the lysozyme of O. savignyi.
Dissertation (MSc (Biochemistry))--University of Pretoria, 2005.
Biochemistry
unrestricted

Indiana University-Purdue University Indianapolis (IUPUI)
The Lyme disease agent, Borrelia burgdorferi, has a complex system that allows it to thrive in the harsh and distinct environments of its tick vector and mammalian host. Although it has been known for some time that the Borrelia oxidative stress regulator protein (BosR) plays a necessary role in mammalian infectivity and functions as a transcriptional regulator of alternative sigma factor RpoS, very little is known about its mechanism of action, other than the suggestion that BosR activates rpoS transcription by binding to certain upstream regions of the gene. In our studies, we performed protein degradation assays and luciferase reporter assays for further understanding of BosR function. Our preliminary findings suggest that BosR is post-transcriptionally regulated by an unknown protease and may not need to bind to any rpoS upstream regions in order to activate transcription. We also describe the construction of luciferase reporter systems that will shed light on BosR’s mechanism of action. We postulate the provocative possibility that unlike its homologs Fur and PerR in other bacterial systems, BosR may not utilize a DNA-binding mechanism in order to fulfill its role as a transcriptional regulator to modulate virulence gene expression.