Abstract Feline immunodeficiency virus (FIV) is an important infectious agent of domestic cats worldwide. It has been classified into the Lentivirus genus of the Retroviridae family, together with human immunodeficiency virus (HIV). Five FIV subtypes (A, B, C, D and E) have been described based on sequence variation of the V3-V5 region of the envelope (env) gene. There is considerable sequence diversity within and between subtypes, which has been a major obstacle in the development of a successful vaccine. However, an FIV vaccine that incorporates inactivated whole viruses from subtypes A and D is now commercially available. Although the vaccine has been shown to be efficacious in protecting against challenge with homologous and a heterologous (subtype B) subtypes, its effectiveness against other viral variants is unknown. Therefore, identifying the type and diversity of FIV strains in different regions is important to establish the potential efficacy of the vaccine in areas where vaccination is to be implemented. The proviral DNA sequence of the V3-V5 region of the env gene was determined for 102 FIV-infected cats from locations in Australia, New Zealand and South Africa. Subtype A was the predominant subtype in Australia and South Africa, although subtype B and C were also identified in each of these countries, respectively. Both subtypes A and C were also present in New Zealand. Of interest, there were some samples in New Zealand and South Africa that demonstrated subtype assignment discrepancies when different regions of the genome were analysed, suggesting co-infection and/or recombination. Cats infected with FIV exhibit varying degrees of immunological impairment. Currently, prognosis for an FIV-infected cat is based on clinical signs alone, which is a relatively subjective measure. In HIV-infected patients it is recognised that viral RNA load correlates with disease stage and prognosis. This PhD research tested whether viral RNA load may be a useful prognostic marker in FIV infection. A real-time PCR assay was developed to quantify plasma viral RNA load in 42 FIV-infected cats at three different clinical stages (1:healthy, 2:unwell without signs of immunodeficiency, 3:unwell with signs of immunodeficiency). In cats older than 5 years of age, log-transformed viral RNA loads were significantly higher in cats in category 3 compared to cats in category 1. There were no significant differences in the viral RNA load of older cats in category 2 compared to category 1. There were no cats younger than 5 years of age in category 3 and there was no significant difference in viral RNA load between young cats in categories 1 and 2. Of the 15 cats for which follow-up data was available, eight showed no change in clinical signs, and seven showed a worsening of clinical signs with six of these showing a progression of clinical category including death. One of the cats in category 2 that progressed clinically had one of the highest viral RNA loads of cats in that category. Three of four cats from category 3 that were followed had either died or been euthanised. Two of these cats had among the highest viral RNA loads in the whole study, while the remaining cat (for which the definitive cause of death was not confirmed) had a relatively low viral RNA load. In summary, measurement of viral RNA load was found to be a potentially useful clinical and prognostic marker but further work is required to better assess its usefulness to veterinarians. Serum acute phase proteins were investigated as possible candidate markers of FIV disease with the aim of developing a more simplified assay that could be used as a prognostic marker for FIV infection. Blood samples from 43 FIV-infected and 25 FIV-negative cats were assayed for the concentration of four acute phase proteins. Both healthy and sick cats were included in the study. Compared to healthy cats, sick cats had significantly higher concentrations of serum amyloid A (P<0.05). Alpha 1-acid glycoprotein and haptoglobin were also found to be in higher concentrations in sick cats (P<0.1). Other variables such as age and gender were also associated with acute phase protein concentrations. With respect to FIV infection, it was found that in sick cats, serum amyloid A, in combination with the age of the cat, was the best predictor of FIV viral RNA load. Alpha 1-acid glycoprotein and haptoglobin were not significantly associated with FIV viral RNA load. Although health status did not influence albumin levels, they were found to be significantly lower in FIV-positive cats in comparison to FIV-negative cats (P<0.05). The frequent monitoring of viral RNA loads and CD4+ lymphocyte counts that is performed on HIV-infected patients is cost prohibitive in veterinary patients. This study showed that there is potential for the use of acute phase protein concentrations (in particular serum amyloid A) as alternative prognostic tools in FIV-infected cats. Further work, particularly longitudinal studies, is required to more definitively define changes in viral RNA load and acute phase protein concentrations throughout the course of FIV infection.

Thesis (MSc (Genetics))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: South Africa is regarded as one of the top wine producing countries in the world. One of the threats to the sustainability of the wine industry is viral diseases of which Grapevine leafroll-associated virus 3 (GLRaV-3) and Grapevine virus A (GVA) are considered to be the most important and wide spread. Scion material is regularly tested for viruses; however scion material is often grafted onto rootstocks that have questionable phytosanitary status. Virus detection in rootstocks is challenging due to low and varying titres, but is imperative as a viral control mechanism. An additional viral control mechanism is the use of transgenic grapevine material which offers resistance to grapevine infection. The objective of this project was to establish a detection system using real time PCR (qPCR) techniques, to accurately and routinely detect GLRaV-3 and GVA in rootstock propagation material. qPCR would furthermore be used to perform molecular characterisation of transgenic plants containing a GLRaV-3 antiviral ΔHSP-Mut construct. A severely infected vineyard (Nietvoorbij farm) in the Stellenbosch area was screened throughout the grapevine growing season to investigate virus prevalence throughout the season and to determine the optimal time for sensitive virus detection. A large scale screening of nursery propagation material for GLRaV-3 infection was also conducted. The qRT-PCR results were compared to DAS-ELISA results to compare the efficacy and sensitivity of the two techniques. For the severely infected vineyard, the ability to detect GLRaV-3 increased as the season progressed towards winter. qRT-PCR was more sensitive and accurate in detecting GLRaV-3 than DASELISA, as the latter technique delivered numerous false positive results later in the season. The best time to screen for GLRaV-3 in the Western Cape region was from the end of July to September. For the nursery screenings, our qRT-PCR results were compared to the results of the DAS-ELISA performed by the specific nurseries. No GLRaV-3 infection was detected in the specific samples received from the two different nurseries. The results for all the samples correlated between the two techniques. This confirms that the propagation material of these nurseries has a healthy phytosanitary status with regards to GLRaV-3. However, the detection of GVA in the severely infected vineyard yielded inconsistent results. Detection ability fluctuated throughout the season and no specific trend in seasonal variation and virus titre fluctuation could be established. The highest percentage of GVA infected samples were detected during September, April and the end of July. Previously published universal primers were used for the detection of GVA, but further investigation indicated that they might not be suitable for sensitive detection of specific GVA variants present in South Africa. Vitis vinifera was transformed with a GLRaV-3 antiviral construct, ΔHSP-Mut. SYBR Green Real time PCR (qPCR) and qRT-PCR were utilised as alternative methods for molecular characterisation of transgenic plants. The qPCR and Southern blot results correlated for 76.5% of the samples. This illustrated the ability of qPCR to accurately estimate transgene copy numbers. Various samples were identified during qRT-PCR amplification that exhibited high mRNA expression levels of the transgene. These samples are ideal for further viral resistance studies. This study illustrated that the versatility of real time PCR renders it a valuable tool for accurate virus detection as well as copy number determination. / AFRIKAANSE OPSOMMING: Suid Afrika word geag as een van die top wyn produserende lande ter wereld. Die volhoubaarheid van die wynbedryf word onder andere bedreig deur virus-infeksies. Grapevine leafroll associated virus 3 (GLRaV-3) en Grapevine virus A (GVA) is van die mees belangrike virusse wat siektes veroorsaak in Suid-Afrikaanse wingerde. Wingerd bo-stok materiaal word gereeld getoets vir hierdie virusse, maar hierdie materiaal word meestal geënt op onderstokmateriaal waarvan die virus status onbekend is. Virus opsporing in onderstokke word egter gekompliseer deur baie lae en variërende virus konsentrasies, maar opsporing in voortplantingsmateriaal is ‘n noodsaaklike beheermeganisme vir virus-infeksie. Die doel van die projek was om ‘n opsporingsisteem te ontwikkel via kwantitatiewe PCR (qPCR) tegnieke vir akkurate en gereelde toetsing van GLRaV-3 en GVA in onderstokmateriaal. qPCR sal ook verder gebruik word vir molekulêre karakterisering van transgeniese plante wat ‘n GLRaV-3 antivirale ΔHSP-Mut konstruk bevat. ‘n Hoogs geïnfekteerde wingerd was regdeur die seisoen getoets om seisoenale fluktuasies in viruskonsentrasie te ondersoek en om die optimale tydstip vir sensitiewe virus opsporing te bepaal. ‘n Grootskaalse toetsing van kwekery voortplantingsmateriaal vir GLRaV-3 infeksie was ook uitgevoer. Die qRT-PCR resultate is met die DAS-ELISA resultate vergelyk om die effektiwiteit en sensitiwiteit van die twee tegnieke te vergelyk. Vir die hoogs geïnfekteerde wingerd het die GLRaV-3 opsporing toegeneem met die verloop van die seisoen tot en met winter. qRT-PCR was meer sensitief en akkuraat as DAS-ELISA in die opsporing van GLRaV-3, weens verskeie vals positiewe resultate wat later in die seisoen deur die laasgenoemde tegniek verkry is. Die beste tyd om vir GLRaV-3 te toets is vanaf einde Julie tot September. Tydens die kwekery toetsings was qRT-PCR resultate met die DAS-ELISA resultate van die spesifieke kwekerye vergelyk. Geen GLRaV-3 infeksie was waargeneem in die spesifieke monsters wat vanaf die kwekerye ontvang is nie. Die resultate van die twee tegnieke het ooreengestem vir al die monsters wat v getoets is. Dit het bevestig dat die voortplantingsmateriaal van hierdie kwekerye gesonde fitosanitêre status met betrekking tot GLRaV-3 gehad het. Die opsporing van GVA in die geïnfekteerde wingerd het egter wisselvallige resultate gelewer. Opsporing van die virus het ook regdeur die seisoen gefluktueer en geen spesifieke neiging in seisoenale opsporingsvermoë kon gemaak word nie. Die hoogste persentasie GVA geïnfekteerde monsters was waargeneem tydens September, April en die einde van Julie. Voorheen gepubliseerde universele inleiers was gebruik vir die opsporing van GVA, maar verdere ondersoeke het getoon dat hierdie inleiers nie noodwendig geskik is vir sensitiewe opsporing van GVA variante wat teenwoordig is in Suid-Afrika nie. Vitis vinifera was getransformeer met ‘n GLRaV-3 antivirale konstruct, ΔHSP-Mut. SYBR Green Real time PCR (qPCR) en qRT-PCR was ingespan as alternatiewe metodes vir molekulêre karaterisering van transgeniese plante. Die qPCR en Southern-klad resultate het ooreengestem vir 76.5% van die monsters. Dit illustreer die vermoë van qPCR om akkurate kopie-getalle van transgene te bepaal. Verskeie plante is geïdentifiseer tydens qRT-PCR amplifisering wat hoë vlakke van transgeen mRNA uitdrukking getoon het. Hierdie monsters is ideaal vir verdere virus weerstandbiedendheids studies. Hierdie studie het die veelsydigheid van real time PCR bewys en getoon dat dit ‘n kosbare tegniek is vir akkurate virus opsporing sowel as kopie-getal bepaling.

A Terra Preta Antropogênica (TPA) é considerada um dos solos mais férteis do mundo e recebe essa denominação por ser originada da ação antrópica, provavelmente de populações pré-colombianas que viveram nestes sítios arqueológicos. O crescente aumento por agricultura sustentável torna a utilização de bactérias produtoras de antibióticos uma alternativa de controle para doenças de plantas. Pirrolnitrina (PRN) é um antibiótico que tem ampla atividade antimicrobiana produzida por várias estirpes de Burkholderia e Pseudomonas que foram isoladas de diferentes solos. Entretanto, não se tem conhecimento de isolados bacterianos de TPA que produzem PRN, assim com, sobre a ecologia e freqüência do gene para este antibiótico. A PRN é codificada por um operon composto por 4 genes sendo o gene prnD responsável por catalisar a oxidação que forma a pirrolnitrina. Neste trabalho, foi estudado o gene prnD através de métodos dependente e independente de cultivo. Foram utilizados isolados bacterianos para detectar o gene prnD através de PCR convencional e a identificação feita pelo seqüenciamento. As bactérias com amplificação positiva tiveram suas sequências do gene analisadas no programa MOTHUR o qual reuniu as em 10 grupos. Um representante de cada grupo foi empregado no teste de antagonismo contra o fitopatógeno Fusarium oxysporum. Amostras de solo de TPA e seus solos adjacentes foram coletados de dois sítios: Caldeirão Capoeira (floresta secundária por mais de 20 anos) e Cultivado (cultivado com mandioca por pelo menos 30 anos). Os DNAs totais das amostras de solo extraído foram usados como molde nas reações de PCR quantitativo em tempo real para verificar a abundância do gene prnD nas amostras de solo. Em amostras de solo também foi quantificado o gene 16S rRNA. No estudo dependente de cultivo, do total de 219 isolados (175 Burkholderia e 44 Pseudomonas), 60 isolados do gênero Burkholderia e 3 de Pseudomonas exibiram amplificação positiva. A análise filogenética do gene prnD mostrou que a maioria das seqüências obtidas deste estudo não agruparam com as seqüências do banco de dados do GenBank indicando que há diversidade do gene prnD nos isolados de solos amazônicos e que podem ser distintos dos descritos anteriormente. O teste de antagonismo demonstrou que isolados com potencial genético para produção de pirrolnitrina também são bioativos contra Fusarium oxysporum, apresentando forte atividade antimicrobiana. No estudo independente de cultivo, no sítio Caldeirão Cultivado, solo de TPA apresentou maior número de cópias do gene prnD e do gene 16S rRNA em relação ao solo ADJ. No sítio Caldeirão Capoeira, o solo adjacente apresentou maior quantidade do gene prnD (1,74x105 cópias/g solo) que o solo de TPA (2,48x104 cópias/ g de solo), no entanto, na TPA as bactérias totais foram mais abundante. Estes resultados evidenciam que a metodologia de PCR quantitativo em tempo real desenvolvida foi altamente sensível e específica permitindo a detecção de diferenças sensíveis e significativas entre os solos na quantificação do gene prnD. A abundância do gene prnD correlacionou significativamente com parâmetros químicos do solo tais como pH, fósforo, cálcio, magnésio e micronutrientes / Anthropogenic Dark Earth (ADE) is considered one of the world\'s most fertile soils and receives this name because it originated from human action, probably by pre-Columbian populations who lived in these archaeological sites. Because of the common trend in agriculture towards sustainability antibiotic-producing bacteria is an alternative for biocontrol to plant diseases. Pyrrolnitrin (PRN) is a broad-spectrum antibiotic produced by various strains of Burkholderia and Pseudomonas that have been isolated from different soils. However, little is known about PRN-producing bacteria screened from ADE, even as the ecology and frequency of pyrrolnitrin gene. PNR is encoded by an operon comprised by four genes and prnD gene is responsible for catalyze the oxidation to form pyrrolnitrin. In this work, we studied the gene prnD through culture dependent and independent methods. Conventional PCR were established to detect prnD gene in bacterial isolates screened in ADE and their adjacent soils (ADJ) and identification were done by sequencing. Based on the results generated by MOTHUR prnD sequences from isolates were grouped into 10 groups. A representative of each group was used in the test of antagonism against the plant pathogen Fusarium oxysporum. Soil samples of ADE and its adjacent soils were collected from two sites: Caldeirão Capoeira (secondary forest for over 20 years) and Caldeirão Cultivado (cultivated with cassava for at least 30 years). The total DNA extracted from soil samples was used as template in quantitative PCR reactions to determine the abundance of prnD gene. In soil samples was also quantified the 16S rRNA. In the study culture-dependent, in a total of 219 isolates (175 Burkholderia and 44 Pseudomonas), 60 isolates of Burkholderia and 3 Pseudomonas exhibited positive amplification for prnD gene. Phylogenetic analysis of prnD gene showed that most of the sequences obtained in this study grouped distinctly from sequences of the GenBank database. It indicates that there is diversity in prnD gene of isolates from amazonian soils and they may differ from those previous described. The antagonism test showed that isolates with genetic potential for production of pyrrolnitrin are also bioactive against Fusarium oxysporum, exhibiting strong antimicrobial activity. In culture-independent study, the site Caldeirão Cultivado, ADE soil showed higher copies number of prnD gene and 16S rRNA gene than in ADJ soil. At the site Caldeirão Capoeira, surrounding soil had a higher amount of prnD gene (1.74 x105 copies / g soil) than ADE soil (2.48 x104 copies / g soil), however, in ADE total bacteria was more abundant. These results show that the real-time PCR assay developed in this study was highly sensitive and specific enabling the detection of sensitive and significant differences between soils in the quantification of prnD gene. Soil variables such as pH, phosphorus, calcium, magnesium and micronutrients significantly correlated with abundance of prnD gene

A combinação do aumento da população mundial e a melhoria nos padrões de vida estão levando a uma maior demanda por alimentos com alto valor nutricional. As variedades de laranjas de polpa vermelha são as únicas laranjas que acumulam licopeno na polpa e no suco. O licopeno é um carotenóide considerado como um composto antioxidante, com ação potencial na prevenção de alguns tipos de câncer. Apesar do valor nutricional agregado as laranjas de polpa vermelha, a caracterização fisiológica e molecular dos carotenóides nessas laranjas permanece pouco estudada. O objetivo deste trabalho foi caracterizar o perfil de transcrição de genes envolvidos na via de biossíntese dos carotenóides e quantificar a concentração dos carotenóides na polpa dos frutos de duas laranjas de polpa vermelha e da laranja Pêra (controle). Para comparação entre variedades, os frutos das laranjas Sanguínea-de-Mombuca (\'SM\') e Bahia Cara-Cara (\'CC\'), que possuem polpa vermelha, e da laranja Pêra (\'P\') foram colhidos aos 270, 300 e 330 dias após o florescimento (DAF), em Cordeirópolis (CCSM). Para identificar a influência climática no acúmulo de carotenóides, frutos da laranja de polpa vermelha \'SM\' foram também colhidos frutos aos 270, 300 e 330 DAF em diversos locais: CCSM, Mogi-Mirim (MM) e São Bento do Sapucaí (SBS). A quantidade dos transcritos dos genes fitoeno sintase (PSY), fitoeno dessaturase (PDS), \'dzeta\'-caroteno dessaturase (ZDS), carotenóide isomerase (CRTISO), licopeno \'épsilon\'-ciclase (LCY\'\'épsilon\'), licopeno \'beta\'-ciclase (LCY\'beta\'), hidroxilase \'beta\'-caroteno (HY\'beta\') e zeaxantina epoxidase (ZEP), que codificam para as enzimas da via de biossíntese dos carotenóides, foram analisados por PCR em tempo real. A quantificação dos principais carotenóides presentes no suco dos frutos foi realizada por meio de cromatografia líquida de alta eficiência. A análise quantitativa de transcritos obtidos a 330 DAF indicaram a existência de maiores níveis de expressão dos genes precursores dos carotenos (PSY, PDS, CRTISO, ZDS, LCY\'beta\') nas variedades de polpa vermelha, principalmente na \'SM\'. A quantidade dos carotenos fitoflueno, licopeno e \'beta\'-caroteno foram maiores nas laranjas vermelhas, em relação ao observado nos frutos de laranja Pêra, correlacionando com os níveis de expressão gênica. Nos frutos da laranja pigmentada \'SM\' coletadas aos 330 DAF pôde-se observar uma possível associação entre o clima do local de cultivo e o conteúdo de transcritos dos genes da via de carotenóides. O cultivo em clima mais quente (MM) resultou em frutos com maiores níveis de transcritos dos genes precursores de carotenos (PSY, PDS, ZDS, CRTISO e LCY\'beta\'), enquanto que o cultivo em região de clima mais frio (SBS) resultou no aumento do nível de transcritos dos genes precursores das xantofilas (HY\'beta\' e ZEP). Como consequência, houve um acúmulo maior dos carotenos fitoflueno, licopeno e \'beta\'-caroteno, nos frutos cultivados em locais quentes, porém, em regiões mais frias, houve maior acúmulo das xantofilas zeaxantina e violaxantina, nos frutos. Com o sequenciamento das regiões de diferentes genes da via de biossíntese de carotenóides, foram identificadas algumas mutações pontuais na sequência de nucleotídeos dos genes PSY, CRTISO e ZDS da variedade pigmentada \'SM\', em comparação com os mesmos genes da laranja Pêra. Algumas dessas mutações resultaram na alteração de aminoácidos na sequência das proteínas e deste modo, supõe-se que estes genes sejam os principais candidatos a serem os responsáveis pela alteração do fenótipo das variedades que produzem frutos com polpa avermelhada, ricos em licopeno / The increasing world population and improvement in living standards are leading to greater demand for foods with high nutritional value. The sweet orange varieties with red-flesh are the only ones that accumulate lycopene in the pulp and juice. Lycopene is a carotenoid compound with antioxidative property which may have a potential action in preventing some cancers. Despite of the high nutritional value in the red-fleshed oranges, the physiological and molecular characterization of carotenoids remains to be elucidated. The present work aimed to characterize the transcriptional profile of genes involved in carotenoids biosynthesis pathway and quantify the carotenoids in red-fleshed oranges and Pêra orange (as a control). For a direct comparison among the varieties, fruits of Sanguínea-de-Mombuca (\'SM\') and Bahia Cara-Cara (\'CC\') red-fleshed sweet oranges, and Pêra orange were harvested at 270, 300 and 330 days after flowering (DAF) in Cordeirópolis station (CCSM). To investigate the impact of climate variation on carotenoids accumulation, red-fleshed fruits \'SM\' orange were harvested on 270, 300 and 330 DAF in different climate locations: Cordeirópolis station (CCSM), Mogi-Mirim (MM) and São Bento do Sapucaí (SBS). Quantitative transcriptional analysis of the genes phytoene syntase (PSY), phytoene desaturase (PDS), \'dzeta\'-carotene desaturase (ZDS), carotenoid isomerase (CRTISO), lycopene \'épsilon\'-ciclase (LCY\'épsilon\'), lycopene \'beta\'-cyclase (LCY\'beta\'), hydroxylase \'beta\'-carotene (HY\'beta\') and zeaxanthin epoxidase (ZEP) related to the carotenoids biosynthesis pathway were performed by quantitative Real Time PCR. Further, carotenoids quantification in red-fleshed fruits was measured by high performance liquid chromatography. The transcriptional profile at 330 DAF revealed high expression levels for the carotene gene precursors (PSY, PDS, CRTISO, ZDS, LCY\'beta\') in red-fleshed oranges, in particular to \'SM\' orange when compared to Pêra orange. Carotenoids profiling displayed higher phytofluene, lycopene and \'beta\'-carotene concentrations in red-fleshed orange than Pêra orange fruits. Transcript levels of genes related to the carotenoids pathway were responsive to the climate variation in red-fleshed orange harvested at 330 DAF. In warmer climate conditions, increased expression levels were found for the genes related carotene precursors (PSY, PDS, ZDS, CRTISO and LCY\'beta\'), whereas under colder climate conditions, the red-fleshed fruits orange showed higher expression levels for the xanthophylls precursors genes (HY\'beta\' e ZEP). As a consequence, the carotenes phytofluene, lycopene and \'beta\'-carotene accumulated in warmer climate developed fruits, instead of colder climate conditions, where the fruits accumulate increased concentrations of xanthophylls such as zeaxanthin and violaxanthin. Moreover, the sequencing of fragments from different genes related to the carotenoids pathway enabled the identification of point mutations for the genes PSY, CRTISO and ZDS in the red-fleshed variety. These mutations caused amino acid changes in the protein which indicates that these genes might be responsible for the phenotypes red-fleshed fruits and increased lycopene levels shown in the variety \'SM\'

Made available in DSpace on 2014-06-11T19:32:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-01-28Bitstream added on 2014-06-13T20:44:13Z : No. of bitstreams: 1 oliveira_e_dr_jabo.pdf: 277356 bytes, checksum: 7f7e43415afd7264c79b023acd4b37ac (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O presente estudo foi realizado com o intuito de desenvolver o PCR quantitativo para detecção dos níveis de mRNA da toxina botulínica tipo A em palmito. Foram desenhados primers para o gene codificador da toxina botulínica tipo A, os quais apresentaram especificidade e sensibilidade elevadas. Cepas referência de Clostridium botulinum tipo B, C, D, E, F, G e Clostridium butyricum, baratii e argentinense foram utilizadas para verificação da especificidade dos primers sintetizados. Diluições seriadas de cultura da cepa referência de Clostridium botulinum tipo A foram inoculadas em amostras de palmito e separadas em dois grupos: palmito comercial com conservantes e palmito sem conservantes e incubadas a 4°, 25° e 37°C. Alíquotas dessas amostras contaminadas experimentalmente foram submetidas a extração de RNA, para determinação do limiar de detecção da técnica de PCR quantitativo, assim como inoculadas em camundongos para o teste de toxicidade aguda (bioensaio). O bioensaio apresentou sensibilidade característica e detectou a presença da toxina botulínica nas amostras de palmito sem conservantes. No entanto, o PCR quantitativo, apresentou sensibilidade maior que aquela observada no bioensaio, detectando a expressão do gene para BoNT/A (botulinum neurotoxin type A) no tratamento de palmito sem conservantes e no tratamento de palmito comercial incubado a 37°C. Isto evidencia a possível utilização desta técnica no diagnóstico de contaminação de amostras de alimento por C. botulinum tipo A. / The present study was realized in order to develop the quantitative assay for the detention of the levei mRNA of the botulinic toxin type A production in palm heart. Primers were designed for the gene encoding the botulinic toxin type A, which showed high specificity and sensitivity. Reference of strains of C. botulinum type S, c, D, E, F, G, C/ostridium butyricum, baratii and argentinense were used for verification of the specificity of the primers synthesized. Serial dilutions culture of the reference strain of C/ostridium botulinum type A were inoculated in the palm heart samples and separated in two groups: commercial palm heart (with preservatives) and palm heart without preservatives and incubated at 4°, 25° and 37°C. Aliquots these contaminated experimentally samples were subjected to extraction of RNA, for determination of the threshold of detection of the technique of quantitative PCR, well as inoculated into mice to test the acute toxicity (bioassay). The bioassay presented its characteristic sensitivity and detected the presence of the botulinic toxin in samples of palm heart without preservatives. However, the quantitative PCR presented greater sensibility than that observed in the bioassay, detecting the expression of the gene for SoNT/A (botulinic neurotoxin type A) in the treatment of palm heart without preservatives and treatment of commercial palm heart incubated at 37°C. This highlights the possible use of this technique in the diagnosis of contamination of samples the food by C. botulinum type A.

Dans ce travail, nous nous sommes intéressés à l’étude des dirofilarioses chez le réservoir canin « le chien » ainsi que chez les vecteurs « moustiques », en particulier, en ce qui concerne la détection, la surveillance et la prophylaxie. Le premier objectif était de développer une PCR duplex en temps réel ciblant le gène COI capable de détecter et de différencier simultanément D. immitis et D. repens. Ainsi, nous avons détecté par cet outil moléculaire, pour la première fois en France, D. immitis et D. repens chez des moustiques tigre "Aedes albopictus". Nous avons, de plus, confirmé la présence de l’infection à D. immitis chez les chiens du nord d'Algérie.Le deuxième objectif était d'évaluer l’intérêt de la spectrométrie de masse MALDI-TOF MS pour la détection de changements dans les profils protéiques d'Aedes aegypti infectés expérimentalement avec des nématodes filaires (D. immitis, Brugia malayi et B. pahangi). Les résultats obtenus montrent la capacité du MALDI-TOF MS à différencier des moustiques infectés et non infectés par les filaires. Ainsi, les meilleurs taux de classification correcte obtenus sont 94,1, 86,6, 71,4 et 68,7% pour les non infectés versus ceux infectés, respectivement, par D. immitis, B. malayi et B. pahangi.Le troisième objectif de ce travail était l’évaluation de l'efficacité anti-gorgement et insecticide d'un ectoparasiticide (Vectra® 3D) contenant trois principes actifs : le dinotéfurane, le pyriproxyfène et la perméthrine contre Ae. albopictus, l'une des principales espèces vectrices de Dirofilaria spp. Les résultats ont démontré que le DPP a une efficacité anti-gorgement et insecticide significative contre Ae. albopictus / In this work, we are interested in studying dirofilarial infections in dogs and vectors “Mosquitoes” especially detection, monitoring and prophylaxis. The first objective is to develop a real-time duplex PCR targeting the COI gene capable of simultaneously detecting and differentiating D. immitis and D. repens. Subsequently, we applied this tool to a canine dirofilariosis surveillance process in different endemic areas of Mediterranean basin (Corsica and Algeria). We have thus detected by this molecular tool for the first time in France, D. immitis and D. repens in Aedes albopictus mosquitoes. We reported, also, the presence of D. immitis in dogs from northern Algeria.The second aim was to assess whether the MALDI-TOF MS can detect changes in the protein profiles of Aedes aegypti infected experimentaly with filarial nematodes (D. immitis, Brugia malayi and B. pahangi). Obtained results showed the potential of MALDI-TOF MS as a reliable tool for differentiating non-infected and filariae-infected Ae. aegypti mosquitoes with a best correct classification rate obtained from the thorax-head part with 94.1 and 86.6, 71.4 and 68.7% for non-infected and D. immitis, B. malayi and B. pahangi infected mosquitoes respectively.The third aim of this work has focused on the evaluation of the anti-feeding and insecticidal efficacy of an ectoparasiticide (Vectra® 3D) containing three active ingredients: dinotefurane, pyriproxyfen and permethrin (DPP) against Ae. albopictus. Results demonstrated that the DPP combination has significant anti-feeding and insecticidal efficacy against Ae. albopictus for at least 4 weeks.

Orientador: Hélio José Montassier / Banca: Adolorata Aparecida Bianco Carvalho / Banca: Clarice Weins Arns / Banca: Geraldo Aleixo da Silva Passos Júnior / Banca: Liana Brentano / Resumo: As respostas imunes inatas e adquiridas, incluindo-se aí tanto as mediadas por fatores humorais como celulares normalmente induzidas após a infecção ou vacinação com o vírus da BI (VBI), são caracterizadas por sua grande complexidade e por aspectos relevantes que ainda são pouco conhecidos, no que tange aos elementos capazes de exercer uma ou mais ações efetoras contra esse patógeno e que culminassem na restrição da replicação viral, seguido de sua eliminação do organismo hospedeiro e também no impedimento de lesões mais severas. Isso posto, foi formulado o presente estudo com o fito principal de fazer a avaliação das respostas imunes humorais e celulares em diferentes intervalos pós-desafio com o VBI de aves previamente vacinadas ou não, realizando-se a mensuração de anticorpos no soro e na lágrima, e a quantificação da expressão de genes relacionados às respostas imunes na superfície traqueal, a fim de correlacionar tais parâmetros com o estado de proteção ao desafio. Os resultados demonstraram que os aumentos significativos nos níveis de anticorpos lacrimais dos isótipos IgG e IgA nas aves previamente vacinadas e também na expressão dos genes relacionados às respostas imunes, sobretudo o CD8, a Granzima A e o IFNg foram correlacionados negativamente com um ou mais parâmetros de alterações patológicas traqueais. Constatou-se também, que a memória das respostas imunes humorais e cito-mediadas conferida por uma única vacinação contra a BI no primeiro dia de idade é dependente da dose vacinal administrada / Abstract: Avian infectious bronchitis virus (IB) is a worldwide infectious disease which causes significant economic losses in poultry industry. The innate and acquired immune responses, including whether there mediated by both cellular and humoral factors that are induced after infection or vaccination with IB virus (IBV) are characterized by their higher complexity and for the relevant aspects that are still poorly known, with respect to the elements able to exercise one or more actions against the pathogen and that culminate in the restriction of its propagation and also on your clearance of the host organism. So, this project was formulated with the main done to make the evaluation of cellular and humoral immune responses at different intervals post-immunization or postchallenge with IBV poultry previously vaccinated or not, performing the measurement of antibodies in serum or tears and quantitation of the expression of genes related to immune responses in tracheal surface, correlating these parameters with the protection against IBV. The results showed that both significative increase of IgG and IgA isotypes in tears of previously vaccinated chickens and the expression levels of genes related to immune responses, especially CD8, Granzyme A and IFN g were negatively correlated with one or more parameters of pathological lesions at trachea. Moreover, we found that the immune memory conferred by vaccination against BI on the first day of age is dependent on vaccine dose administered / Doutor

La néphropathie à BK virus (BKV) est l'une des complications les plus fréquentes de la transplantation rénale. La prise en charge consiste en la réduction préemptive de l'immunosuppression basée sur le suivi de la charge virale, mais cette stratégie n’est pas complètement efficace et augmente le risque de rejet. Dans un premier volet, nous avons évalué la mesure de la charge virale par PCR quantitative en temps réel, permettant de mettre en évidence des facteurs de variabilité comme le polymorphisme du BKV et de valider la technique utilisée pour le suivi de notre cohorte. L’intérêt des anticorps neutralisants (AcNs) anti-BKV en tant que marqueur prédictif de la réplication BKV a ensuite été évalué dans une cohorte de 168 transplantés rénaux. Nous avons montré i) que le virus responsable de l’infection provenait du donneur ; ii) que les AcNs jouent un rôle dans la prévention de la réactivation et le contrôle de la réplication virale et iii) qu’un seuil d’AcNs de 4 log10 permettait de stratifier le risque de réplication BKV. Ce travail ouvre la voie à un suivi personnalisé en fonction du risque de réplication BKV et à de nouvelles approches immunothérapeutiques. / BK virus (BKV)-associated nephropathy is one of the major causes of graft dysfunction and loss in kidney transplant recipients. Since no BKV-specific antiviral therapies are available, management relies on preemptive immunosuppression reduction based on viral load monitoring. However, this strategy does not fully eliminate the risk of nephropathy and can increase the risk of graft rejection. In this work, we evaluated viral load measurement by quantitative real-time PCR in an interlaboratory comparison. Variability factors such as BKV polymorphism or pre-PCR steps have been highlighted and the method used for monitoring our cohort has been validated. The role of anti-BKV neutralizing antibodies (NAbs) as a predictive marker of BKV replication has been investigated in a cohort of 168 kidney transplant recipients. We showed that i) viral infection is caused by the donor strain; ii) NAbs play an essential role in viral replication prevention and control and iii) a NAbs cutoff of 4 log10 allows to stratify BKV replication risk. This work paves the way for personalized monitoring according to BKV replication risk and for new preventive or therapeutic strategies.

O Brasil é um grande produtor de alimentos e a bovinocultura tem grande importância econômica e social. Doenças virais podem ter grande impacto na pecuária e, por isso, a identificação do agente, o conhecimento de sua epidemiologia e o desenvolvimento de técnicas eficientes para seu diagnóstico são medidas extremamente importantes no seu controle. A diarréia viral bovina (“bovine viral diarrhea”- BVD) é uma enfermidade difundida nos rebanhos bovinos ocasionando grandes perdas econômicas em rebanhos de corte e leite em todo mundo. A identificação de animais persistentemente infectados (PI) pelo vírus da BVD (BVDV) é essencial em um programa de controle desta doença. No presente estudo, foi estabelecida uma transcrição reversa seguida da reação em cadeia da polimerase quantitativa (RT-qPCR) específica para identificação e quantificação dos diversos genótipos de pestivírus. Foi utilizado o teste de imunoperoxidase (IPX) como referência, além de outros testes de diagnóstico, como transcrição reversa seguida da reação em cadeia da polimerase (RTPCR), isolamento viral (IV), ELISA de captura de antígeno (ECA) e imunohistoquímica (IHQ). Os valores usados como base para quantificação foram pré-determinados pelo resultado da IPX de uma amostra padrão. A sensibilidade da RT-qPCR foi de 103,9 TCID50/mL, apresentando valor de coeficiente de correlação de 0,978 que permite identificar 95% das infecções com até 450,66 partículas virais/mL de soro. O coeficiente de variação da reprodutibilidade variou de 4 a 9%. Foram analisadas 72 amostras de soro sanguíneo de animais suspeitos de apresentarem infecção persistente pelo BVDV. Os títulos obtidos através da IPX a partir de soro de animais PI variaram de 105,55 a 107,3 TCID50/mL, enquanto que os títulos das amostras testadas pela RTqPCR variaram de 106,2 a 107,6 TCID50/mL. Os diferentes métodos laboratoriais utilizados para detectar o BVDV em rebanhos apresentaram a mesma eficiência entretanto, a utilização comparativa destes métodos aliada a uma nova estratégia permitiu que se estabelecesse uma RT-qPCR específica para detecção e quantificação do BVDV em amostras de soro de animais PI que poderá ser utilizada como uma importante ferramenta para o diagnóstico de pestivírus com diferentes características genéticas. Além disso, o presente trabalho poderá servir como base para a quantificação do agente em diferentes órgãos dos animais PI, o que contribuirá para o entendimento da patogenia do BVDV. / Brazil is an important food producer and cattle production has great economical and social importance. Viral diseases are constant problems and the identification of the agent, the knowledge of the disease´s epidemiology and the development of efficient diagnostic techniques are extremely important for its control. Bovine viral diarrhea (BVD) is a widespread disease causing great economical losses around the world. The identification of persistently infected animals (PI) in bovine herds is essential to the implementation of control programs. In the present study, a reverse transcription followed by quantitative polymerase chain reaction (RT-qPCR) was established to the identification and quantification of pestivirus. Immunoperoxidase (IPX) was used as the reference test, in addition with reverse transcription followed by the polymerase chain reaction (RT-PCR), virus isolation (VI), antigen-capture ELISA (ACE) and Immunohistochemistry (IHC). The values used as standard to quantification were predetermined by the result from the IPX using a reference strain. The sensitivity of RTqPCR was 103.9 TCID50/mL, showing 0.978 of coefficient correlation, what allowed to detect 95% of infections with up to 450.66 viral particles/mL. The reproducibility of the coefficient of varied from 4 to 9%. Seventy-two samples of blood serum from animals suspected of presenting persistent infection of BVDV were analyzed. The titers obtained through the IPX from the serum of PI animals varied from 105.55 to 107.3 TCID50/mL, while the titers from the tested samples by the RT-qPCR varied from 106.2 to 107.6TCID50/mL. The different laboratory methods tested were capable of detecting the BVDV with equal efficiency, The comparative utilization of these methods allied with a new strategy allowed the establishment of a specific RT-qPCR protocol to quantify and detect the BDVD in PI sera which can be used as an important tool to the detection and quantification of the pestivirus different genetic backgrounds. Beyond this, the present protocol can be used to quantify the virus in different organs of PI animals and will contribute for the understanding of BVDV patogenicity.

Introdução: O gênero Acinetobacter representa um importante patógeno relacionado a infecções hospitalares, principalmente, à pneumonia associada à ventilação mecânica (PAV). O aumento de isolados de Acinetobacter baumannii resistentes aos carbapenêmicos (ABRC) representa um problema mundial, pois limita drasticamente as opções terapêuticas. A produção da carbapenamase OXA-23, uma β-lactamase da classe D de Ambler, representa o principal mecanismo responsável por esta resistência em nosso país. Objetivo: O presente estudo tem como objetivo padronizar a técnica de PCR em tempo real (qPCR) para a detecção do gene blaOXA-23 diretamente de aspirados traqueais (ATs) de pacientes com suspeita de pneumonia associada à ventilação mecânica (PAV) de unidades de tratamento intensivo (UTIs). Métodos e resultados: O DNA das amostras de ATs coletadas de pacientes em ventilação mecânica foi analisado pela técnica de qPCR, utilizando o SYBR green, para a detecção do gene blaOXA-23. Dentre os 20 ATs analisados, ABRC foi isolado em 10, A. baumannii sensível aos carbapemêmicos (ABSC) em 3 e 7 foram negativos na culura bacteriológica. O gene blaOXA-23 foi detectado tanto na colônia quanto no AT em 8 das 10 amostras de ABRC. Em uma amostra não houve detecção do gene por qPCR em nenhum dos materiais e em outra amostra houve detecção só no AT. Dos ABSC, em duas amostras não houve detecção do gene na colônia e no AT enquanto que em uma amostra o gene foi detectado somente no AT. Em nenhuma das amostras de ATs negativas na cultura foi detectado o gene. Conclusão: O estudo sugere que a técnica qPCR pode ser aplicada para a detecção do gene blaoxa-23 diretamente de amostras de AT, reduzindo assim, o tempo para o início de uma terpia antimicrobiana adequada e melhorando, consequentemente, o desfecho clínico deste pacientes. / Background: The genus Acinetobacter is an important pathogen associated with nosocomial infections mainly ventilator-associated pneumonia (VAP). The increasing of carbapenemresistant Acinetobacter baumannii (CRAB) isolates is a worldwide concern since it limits drastically the range of therapeutic alternatives. The OXA-23 producing, a carbapenemhydrolysing class D β-lactamase, is the major mechanism responsible for CRAB in our country. Objective: The study objective is to develop a real time PCR (qPCR) to detect the Acinetobacter baumannii blaOXA-23 gene directly in endotracheal aspirate (ETA) of patients under mechanical ventilation, with suspected ventilator-associated pneumonia (VAP). Methods and Results: DNA extracted from ETA samples from patients under mechanical ventilation was analyzed by qPCR, using SYBR green, for the presence of blaOXA-23 gene. Among the 20 ETAs examined, CRAB isolates were recovered in 10 quantitative cultures; carbapenem-susceptible A. baumannii (CSAB) in 3 and 7 cultures were negative to A. baumanni. Of the 10 CRAB, 8 were positive for blaOXA-23 on both the colony and ETA. In one blaOXA-23 qPCR was negative in colony and directly from ETA, while the other showed a qPCR negative result in the colony and positive in the ETA. In 3 CSAB, 2 samples showed negative results in colony and ETA and one showed a blaOXA-23 positive result only from the ETA. None of the 7 negative ETAs were positive for blaOXA-23 gene in the qPCR of the ETA. Conclusion: Our study suggests that qPCR can be applied to detect the presence of blaOXA-23 gene directly from ETAs reducing the time to an earlier initiation of appropriate therapy improving, consequently, the clinical outcomes for these patients.