Greening disease in South Africa is associated with a phloem-limited member of the Alphaproteobacteria known as ‘Candidatus Liberibacter africanus’ (Laf). Spread of this bacterium is assisted by the flight and feeding activities of its triozid vector, Trioza erytreae. In addition to Laf, ‘Candidatus Liberibacter africanus subsp. capensis’ (LafC) has been described from this country. LafC is widely associated with Calodendrum capense, an indigenous Rutaceous species. This dissertation aims to determine (i) whether reservoir hosts exist for Laf amongst indigenous rutaceous species that also act as native hosts to T. erytreae and (ii) whether LafC can be transmitted vertically through Ca. capense seeds. To achieve the first aim, a total of 234 Clausena anisata, 289 Vepris lanceolata and 231 Zanthoxylum capense specimens were assessed for the presence of Liberibacter by making use of a Liberibacter generic real-time PCR assay. Positive samples were further characterized by using a multi-gene phylgenetic approach based on the nucleotide sequences for the 16S, rplJ and omp genes, which respectively encode the 16S ribosomal RNA (rRNA) subunit, 50S ribosomal subunit protein L10 and the outer membrane protein. The tree host species from which Liberibacter sequences were obtained were DNA barcoded using standard procedures. Of the trees tested, 33 Cl. anisata, 17 V. lanceolata, 9 Z. capense and 1 Zanthoxylum davyi, tested positive for Liberibacter. Phylogenetic analysis of the rplJ and omp gene regions, revealed unique Liberibacter clusters associated with each tree species. Phylogenetic analysis of 16S data indicated that the strains detected in V. lanceolata and Cl. anisata were grouped with LafC, while those from Zanthoxylum species grouped separately. The presence of these bacteria in indigenous Rutaceous species should be further investigated to determine whether they are capable of being transmitted to and causing disease on commercial citrus species. To address the second aim of the study, seeds of Ca. capense were collected from two mother trees with known LafC infection. Following total DNA extraction from the midribs of leafs grown from these seeds, a LafC directed real-time PCR system was used to detect the bacterium. However, none of the samples had Ct values <35, the positive/negative threshold. From this study, no indication of seed-transmission was obtained within the limited number of samples which could be tested. The lack of vertical transmission of both Laf and LafC is seeminly a characteristic shared amongst African Liberibacter species. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Microbiology and Plant Pathology / unrestricted

Amblyomma maculatum (the Gulf Coast tick), an aggressive, human-biting, Nearctic and Neotropical tick, is the principal vector of Rickettsia parkeri in the United States. This pathogenic spotted fever group Rickettsia species has been identified in 8-52% of questing adult Gulf Coast ticks in the southeastern United States. To our knowledge, R. parkeri has not been reported previously from adult specimens of A. maculatum collected in Kansas or Oklahoma. A total of 216 adult A. maculatum ticks were collected from 18 counties in Kansas and Oklahoma during 2011-2014 and evaluated by molecular methods for evidence of infection with R. parkeri. No infections with this agent were identified; however, 47% of 94 ticks collected from Kansas and 73% of 122 ticks from Oklahoma were infected with ". Candidatus Rickettsia andeanae" a spotted fever group Rickettsia species of undetermined pathogenicity. These preliminary data suggest that " Ca. R. andeanae" is well-adapted to survival in populations of A. maculatum in Kansas and Oklahoma, and that its ubiquity in Gulf Coast ticks in these states may effectively exclude R. parkeri from their shared arthropod host, which could diminish markedly or preclude entirely the occurrence of R. parkeri rickettsiosis in this region of the United States.

The variation in the insect immune system is an important regulator of insect populations and the pathogens they carry. A central component of insect immunity is melanin, whose production creates cytotoxic intermediates that help to protect against a broad spectrum of pathogens. Melanin is also used in insect cuticle where it helps to improve thermoregulation and desiccation resistance, with insects having less melanized cuticles in warmer and more humid environments. Considering that cuticle melanin and immune melanin are formed by near identical biochemical pathways, they are pleiotropically linked (that is, one or more linked genes influence multiple traits). This has lead to the cuticle-dependent immune investment (CDII) hypothesis, which states that adaptive responses in the cuticle can lead to non-adaptive changes in immunity and could lead to an increase in transmission of insect vectored pathogens in warming climates, due to a weaker defense against the pathogen. However, the impact of CDII on cuticle melanin and immunity, as well as infection prevalence and intensity, under seasonal conditions in the field is still unclear. In this project, we study a population of Asian citrus psyllids, Diaphorina citri, in the field over four seasons. Diaphorina citri vectors a Gram-negative bacteria, Candidatus Liberbacter asiaticus (CLas), that is responsible for Huanglongbing, aka citrus greening disease, which has cost the Florida citrus industry several billion dollars. We assess pathogen load of CLas by quantitative PCR, and assess levels of phenoloxidase activity in the insect hemolymph to measure insect immune function. We assess levels of cuticle melanin. Our results show a significant correlation between temperature, cuticle melanin, and immune function. However, the affect of seasonality on infection prevalence and intensity remains unclear. THE EFFECT OF CLIMATE ON PHYSIOLOGY AND IMMUNE FUNCTION IN THE ASIAN CITRUS PSYLLID, DIAPHORINA CITRI

La Flavescence dorée est une maladie à phytoplasme incurable qui affecte la vigne. Cette pathologie est uniquement transmise de plante à plante par la cicadelle vectrice Scaphoideus titanus. Malgré l'importance de cette maladie, peu d'études ont été menées sur le vecteur. Ce travail de thèse vise à apporter des connaissances sur la biologie et l'écologie de S. titanus mais aussi de manière plus générale sur l'écologie des insectes phytophages. Trois grands thèmes ont été abordés dans ce mémoire : i) les relations entre l'insecte et son environnement à travers l'influence des températures hivernales sur les œufs en diapause, ii) les relations de l'insecte avec sa plante hôte en examinant l'attractivité de cette dernière et le comportement alimentaire du vecteur, iii) les interactions entre congénères par l'étude du comportement d'agrégation des larves. Les résultats obtenus indiquent que 1) des températures froides ne sont pas indispensables à la levée de diapause mais affectent la protandrie en régulant le sex-ratio opérationnel, 2) l'attractivité de la plante hôte est en grande partie liée à sa couleur et il existe des différences de comportement en fonction du stade de développement, 3) les mâles et les femelles n'ont pas le même comportement alimentaire, ce qui pourrait expliquer leur différentes capacités de vection, 4) l'agrégation des larves ne semble pas due à un recrutement actif de leur part et pourrait contribuer à un transfert horizontal de phytoplasme d'insecte à insecte via la plante. A terme, ces connaissances pourront contribuer à l'élaboration de pistes alternatives à la lutte chimique actuelle contre cette cicadelle dans le cadre d'une réduction des intrants. / The Flavescence dorée is an incurable phytoplasma disease of grapevine. This pathology is exclusively transmitted from plant to plant by the leafhopper vector Scaphoideus titanus. Despite the importance of this disease, few studies have been conducted on the vector. This thesis aims to provide knowledge on the biology and ecology of S. titanus but also more broadly on the ecology of phytophagous insects. Three major themes were addressed in this thesis: i) the relationships between the insect and its environment through the influence of winter temperatures on diapausing eggs, ii) the relationships of the insect with its host plant by examining the attractiveness of the plant and the feeding behaviour of the vector, iii) interactions between congeners in the study of the aggregative behaviour of larvae. Our results indicate that 1) cold temperatures are not essential to break the diapause but contribute to protandry through operational sex ratio regulation, 2) the attractiveness of the host plant is largely due to its colour and different instars exhibit differences in behavioural choices, 3) males and females have different feeding behaviour which may explain their different efficiencies at vection, 4) the aggregation of larvae did not seem to result from active recruitment and could contribute to horizontal insect to insect transfer of phytoplasma through the plant. Such knowledge can contribute to the development of alternative S. titanus pest management techniques.

A citricultura se destaca como uma das mais importantes atividades do agronegócio brasileiro, sendo o estado de São Paulo o principal produtor mundial de laranja e o maior exportador de suco concentrado e congelado. O psilídeo dos citros (Diaphorina citri) tornou-se nos últimos anos um dos mais importantes vetores na cultura, devido à transmissão de \'Candidatus Liberibacter asiaticus\' e \'Ca. L. americanus\', bactérias associadas ao huanglongbing (HLB), principal doença da citricultura atual. Uma vez que não há variedades comerciais de citros resistentes ao HLB, seu manejo é baseado no uso de mudas sadias, erradicação de plantas infectadas e pelo controle químico do vetor. No entanto, o custo elevado e os significativos danos ambientais causados pelos inseticidas químicos, associados à possibilidade da seleção de populações de psilídeos resistentes a esses produtos, têm levado à busca por estratégias alternativas de manejo do vetor do HLB que sejam mais adequadas e sustentáveis. Nosso grupo tem tentado contribuir nesse sentido, com a prospecção e caracterização de estirpes de Bacillus thuringiensis (Bt) patogênicos ao psilídeo. Sendo assim, os objetivos deste estudo foram: (a) Confirmar a capacidade endofítica e a patogenicidade de estirpes de Bt em seedlings e mudas de citros com diferentes combinações de copa/porta-enxerto; (b) Determinar a concentração letal (CL50) necessária para ocasionar mortalidade em populações de D. citri; (c) Estudar a interação entre toxinas Cry e receptores de membrana do intestino de ninfas e (d) Avaliar a compatibilidade das estirpes selecionadas com agrotóxicos comumente utilizados na citricultura. Os bioensaios realizados com seedlings e mudas de citros demonstraram que as estirpes de Bt (Cyt1A, Cry2Aa, Cry4A, Cry10, Cry11, S1302, S1450 e S1989) translocaram endofiticamente nas plantas, mantendo sua viabilidade e, em sua maioria, o potencial patogênico para ninfas de D. citri. Para seedlings, os valores de mortalidade passaram de 80% para as estirpes S1302 e S1450. Foram observados esporos e células vegetativas de B. thuringiensis subsp. kurstaki expressando green fluorescent protein (Btk::GFP), visualizados por microscopia de fluorescência, aderidos principalmente aos elementos de vasos e no xilema, obtidas de amostras seccionadas transversal e longitudinalmente, de caule e raiz de seedlings e mudas de laranjeira Pera. A CL50 e CL80 para a estirpe S1302 foi de 4,92 × 104 esporos/mL e 6,63 × 107 esporos/mL, respectivamente. Já para a estirpe S1450, 50% de mortalidade das ninfas testadas foi estimada em 2,19 × 104 esporos/mL, e a CL80 foi de 6,18 × 108 esporos/mL, quando utilizadas suspensões de esporos da bactéria diretamente no substrato de seedlings de laranjeira Pera. Os ensaios de ligação demonstraram que todas as toxinas Cry presentes nas estirpes S1302, S1450 e S1989 (Cry1Aa, Cry1Ab, Cry1Ac, Cry2Aa, Cry2Ab2, Cry1B e Cry11) interagiram com os receptores de membrana intestinal, brush border membrane vesicles (BBMV\'s), obtidos de ninfas de D. citri. Ensaios in vivo evidenciaram que as estirpes S1302 e S1450 mostraram-se compatíveis com todos os agrotóxicos comumente utilizados na citricultura, ainda que os produtos à base de cobre e o inseticida Dimetoato tenham sido deletérios em aplicações diretas na bactéria in vitro. Esses dados evidenciam a possibildade do uso de Bt como bioinseticida no manejo integrado do HLB. / Citriculture is one the most important activities of Brazilian agribusiness, and the State of São Paulo being the world\'s leading orange producer and the largest juice exporter. Asian citrus psyllid - ACP (Diaphorina citri) has become one of the most important vectors in the citriculture in recent years, due to the transmission of \'Candidatus Liberibacter asiaticus\' and \'Ca. L. americanus\', bacteria associated with huanglongbing (HLB), the main citrus disease worldwide. Nowadays there are no commercial citrus varieties resistant to HLB, and its management is based on the use of healthy nursery citrus trees, eradication of symptomatic planst from the orchards, and vector chemical control. However, excessive cost and environmental damage due to application of chemical insecticides associated with the possibility of selection of ACP resistant populations, have led researchers to persue alternative strategies for the management of HLB. Our group has contributed with the screening of Bacillus thuringiensis (Bt) strains with potential against ACP. Therefore, our objectives were: (a) to confirm Bt endophytic translocation in citrus seedlings and nursery trees with different scion-rootstock combinations and to evaluate their pathogenicity against D. citri; (b) to estimate the lethal concentration (LC50 and LC80) to D. citri nymphs; (c) to study the interaction between Cry toxins and brush border membrane vesicle (BBMV) of the midgut D. citri nymphs, and (d) to evaluate the compatibility of pesticides with the selected Bt strains. The bioassays with citrus seedlings and nursery trees demonstrated that the Bt strains (Cyt1A, Cry2Aa, Cry4A, Cry10, Cry11, S1302, S1450 and S1989) translocated from roots to young leaves, maintaining their viability and pathogenicity against D. citri nymphs. For the seedlings, we found mortality values up to 80% for strains S1302 and S1450. Btk::GFP spores and vegetative cells were visualized by fluorescence microscopy in citrus seedlings and nursery trees adhered mainly to vessels and xylem, from roots to stems, in cross-section analyses. LC50 and LC80 for strain S1302 were 4.92 × 104 spores/mL and 6.63 × 107 spores/mL, respectively. For strain S1450, 50% mortality of nymphs tested was estimated at 2.19 × 104 spores/mL, and LC80 was 6.18 × 108 spores/mL, when bacterial spore suspensions were applied to citrus seedlings. Binding assays demonstrated that all Cry toxins present in strains S1302, S1450 and S1989 (Cry1Aa, Cry1Ab, Cry1Ac, Cry2Aa, Cry2Ab2, Cry1B and Cry11) interacted with the BBMV obtained from D. citri nymphs. In vivo assays showed that strains S1302 and S1450 were compatible with all pesticides commonly used in citrus orchards, althoug copper-based pesticides and dimethoate insecticide were incompatible in vitro with Bt strains. Our results demonstrate the potential of using Bt as systemic bioinsecticide in the future in HLB management.

A citricultura se destaca como uma das mais importantes atividades do agronegócio brasileiro, sendo o estado de São Paulo o principal produtor mundial de laranja e o maior exportador de suco concentrado e congelado. O psilídeo dos citros (Diaphorina citri) tornou-se nos últimos anos um dos mais importantes vetores na cultura, devido à transmissão de \'Candidatus Liberibacter asiaticus\' e \'Ca. L. americanus\', bactérias associadas ao huanglongbing (HLB), principal doença da citricultura atual. Uma vez que não há variedades comerciais de citros resistentes ao HLB, seu manejo é baseado no uso de mudas sadias, erradicação de plantas infectadas e pelo controle químico do vetor. No entanto, o custo elevado e os significativos danos ambientais causados pelos inseticidas químicos, associados à possibilidade da seleção de populações de psilídeos resistentes a esses produtos, têm levado à busca por estratégias alternativas de manejo do vetor do HLB que sejam mais adequadas e sustentáveis. Nosso grupo tem tentado contribuir nesse sentido, com a prospecção e caracterização de estirpes de Bacillus thuringiensis (Bt) patogênicos ao psilídeo. Sendo assim, os objetivos deste estudo foram: (a) Confirmar a capacidade endofítica e a patogenicidade de estirpes de Bt em seedlings e mudas de citros com diferentes combinações de copa/porta-enxerto; (b) Determinar a concentração letal (CL50) necessária para ocasionar mortalidade em populações de D. citri; (c) Estudar a interação entre toxinas Cry e receptores de membrana do intestino de ninfas e (d) Avaliar a compatibilidade das estirpes selecionadas com agrotóxicos comumente utilizados na citricultura. Os bioensaios realizados com seedlings e mudas de citros demonstraram que as estirpes de Bt (Cyt1A, Cry2Aa, Cry4A, Cry10, Cry11, S1302, S1450 e S1989) translocaram endofiticamente nas plantas, mantendo sua viabilidade e, em sua maioria, o potencial patogênico para ninfas de D. citri. Para seedlings, os valores de mortalidade passaram de 80% para as estirpes S1302 e S1450. Foram observados esporos e células vegetativas de B. thuringiensis subsp. kurstaki expressando green fluorescent protein (Btk::GFP), visualizados por microscopia de fluorescência, aderidos principalmente aos elementos de vasos e no xilema, obtidas de amostras seccionadas transversal e longitudinalmente, de caule e raiz de seedlings e mudas de laranjeira Pera. A CL50 e CL80 para a estirpe S1302 foi de 4,92 × 104 esporos/mL e 6,63 × 107 esporos/mL, respectivamente. Já para a estirpe S1450, 50% de mortalidade das ninfas testadas foi estimada em 2,19 × 104 esporos/mL, e a CL80 foi de 6,18 × 108 esporos/mL, quando utilizadas suspensões de esporos da bactéria diretamente no substrato de seedlings de laranjeira Pera. Os ensaios de ligação demonstraram que todas as toxinas Cry presentes nas estirpes S1302, S1450 e S1989 (Cry1Aa, Cry1Ab, Cry1Ac, Cry2Aa, Cry2Ab2, Cry1B e Cry11) interagiram com os receptores de membrana intestinal, brush border membrane vesicles (BBMV\'s), obtidos de ninfas de D. citri. Ensaios in vivo evidenciaram que as estirpes S1302 e S1450 mostraram-se compatíveis com todos os agrotóxicos comumente utilizados na citricultura, ainda que os produtos à base de cobre e o inseticida Dimetoato tenham sido deletérios em aplicações diretas na bactéria in vitro. Esses dados evidenciam a possibildade do uso de Bt como bioinseticida no manejo integrado do HLB. / Citriculture is one the most important activities of Brazilian agribusiness, and the State of São Paulo being the world\'s leading orange producer and the largest juice exporter. Asian citrus psyllid - ACP (Diaphorina citri) has become one of the most important vectors in the citriculture in recent years, due to the transmission of \'Candidatus Liberibacter asiaticus\' and \'Ca. L. americanus\', bacteria associated with huanglongbing (HLB), the main citrus disease worldwide. Nowadays there are no commercial citrus varieties resistant to HLB, and its management is based on the use of healthy nursery citrus trees, eradication of symptomatic planst from the orchards, and vector chemical control. However, excessive cost and environmental damage due to application of chemical insecticides associated with the possibility of selection of ACP resistant populations, have led researchers to persue alternative strategies for the management of HLB. Our group has contributed with the screening of Bacillus thuringiensis (Bt) strains with potential against ACP. Therefore, our objectives were: (a) to confirm Bt endophytic translocation in citrus seedlings and nursery trees with different scion-rootstock combinations and to evaluate their pathogenicity against D. citri; (b) to estimate the lethal concentration (LC50 and LC80) to D. citri nymphs; (c) to study the interaction between Cry toxins and brush border membrane vesicle (BBMV) of the midgut D. citri nymphs, and (d) to evaluate the compatibility of pesticides with the selected Bt strains. The bioassays with citrus seedlings and nursery trees demonstrated that the Bt strains (Cyt1A, Cry2Aa, Cry4A, Cry10, Cry11, S1302, S1450 and S1989) translocated from roots to young leaves, maintaining their viability and pathogenicity against D. citri nymphs. For the seedlings, we found mortality values up to 80% for strains S1302 and S1450. Btk::GFP spores and vegetative cells were visualized by fluorescence microscopy in citrus seedlings and nursery trees adhered mainly to vessels and xylem, from roots to stems, in cross-section analyses. LC50 and LC80 for strain S1302 were 4.92 × 104 spores/mL and 6.63 × 107 spores/mL, respectively. For strain S1450, 50% mortality of nymphs tested was estimated at 2.19 × 104 spores/mL, and LC80 was 6.18 × 108 spores/mL, when bacterial spore suspensions were applied to citrus seedlings. Binding assays demonstrated that all Cry toxins present in strains S1302, S1450 and S1989 (Cry1Aa, Cry1Ab, Cry1Ac, Cry2Aa, Cry2Ab2, Cry1B and Cry11) interacted with the BBMV obtained from D. citri nymphs. In vivo assays showed that strains S1302 and S1450 were compatible with all pesticides commonly used in citrus orchards, althoug copper-based pesticides and dimethoate insecticide were incompatible in vitro with Bt strains. Our results demonstrate the potential of using Bt as systemic bioinsecticide in the future in HLB management.

L’analyse du microbiote oral et de son évolution séculaire se fait principalement à partir de l’analyse du tartre dentaire ancien des populations passées et du biofilm dentaire des populations modernes. Nous avons dans un premier temps fait le point des connaissances sur la paléomicrobiologie des bactéries et des archaea contenues dans le tartre dentaire et montré que les archaea faisaient partie du microbiote oral chez l'homme. Dans la deuxième partie, nous avons mis en évidence le répertoire des archaea méthanogènes vivant dans la cavité orale par la culture (une nouvelle espèce Methanobrevibacter massiliense, Methanobrevibacter smithii et Methanobrevibacter oralis). La prévalence de ces archaea était significativement plus élevée chez les patients atteints de parodontite que chez les personnes contrôles. Ensuite, nous avons développé une méthode de génotypage Multispacer Sequence Typing pour typer M. oralis et M. smithii et révélé différents génotypes. Enfin, nous avons analysé le répertoire des archaea méthanogènes dans des échantillons de tartre dentaire ancien datant du 14ème au 19ème siècle. La prévalence et la diversité des archaea méthanogènes dans la cavité orale ont diminué significativement au cours des sept derniers siècles. Des archaea méthanogènes ont été retrouvées dans 75% des prélèvements de tartre dentaire (Candidatus M. massiliense à 44,6%, M. oralis à 19,6%, Methanomassiliicoccus luminyensis-like à 12,5%, Candidatus Nitrososphaera evergladensis-like dans un prélèvement et Methanoculleus bourgensis dans un autre prélèvement). Un prélèvement de tartre positif pour Candidatus M. massiliense a été documenté par hybridation in situ en fluorescence. / The analyses of oral microbiome and its secular evolution mainly use dental calculus in past populations and dental plaque in modern populations. In our thesis, we initially reviewed the knowledge actual about bacteria and archaea paleomicrobiology of the dental calculus. The review disclosed that archaea taked part in the secular core-microbiota in past and modern populations. In the second work, we demonstrated the repertoire of methanogenic archaea currently living in the oral cavity using culture-based approach and succeeded in isolating for the first time a new species named Methanobrevibacter massiliense in addition to Methanobrevibacter smithii and Methanobrevibacter oralis from dental plaque in periodontitis patients. This work showed that the prevalence of methanogens was significantly higher in periodontitis patients than in controls. Some methanogenic archaea were involved in periodontitis. Then, we developed Multispacer Sequence Typing to evaluate M. oralis and M. smithii and revealed different genetic variants in these archaea. Finally, we examined the repertory of methanogenic archaea in ancient dental calculus dating from the 14th to the 19th century. The prevalence and diversity of methanogenic archaea in the oral cavity decreased significantly during the last seven centuries. Methanogenic archaea were found in 75% of dental calculis (Candidatus M. massiliense, 44.6%; M. oralis, 19.6%; Methanomassiliicoccus luminyensis-like, 12.5%; Candidatus Nitrososphaera evergladensis-like in one and Methanoculleus bourgensis in one specimen). One Candidatus M. massiliense dental calculus was further documented by fluorescent in situ hybridization.

Globally, vector borne diseases cause more than a million deaths each year and more than a billion infections in humans. Ticks are of big medicinal importance since they can transmit pathogens that can cause serious infections. Some recently discovered pathogens that can cause infections in humans are Anaplasma phagocytophilum (A. phagocytophilum) that can cause human granulocytic anaplasmosis (HGA) and Candidatus Neoehrlichia mikurensis (N. mikurensis) that can cause Neoehrlichiosis. It is still widely unknown how prevalent these pathogens are, if ticks can be infected with both of these pathogens and if these pathogens can be transovarially transmitted from adult female to egg and larvae. This study aims to screen for these pathogens in collected ticks from southern Sweden and to detect eventual co-infections and transovarial transmission. A real-time qPCR assay targeting the 16S rRNA gene of N. mikurensis and other Anaplasmataceae was applied on 1356 Ixodes ricinus (I. ricinus) ticks collected from 5 sites in southern Sweden. Positive samples were subjected to Sanger sequencing. A. phagocytophilum occurred in 4.64 % of the ticks, N. mikurensis occurred in 1.33 % of the ticks and also Rickettsia was found to occur in 6.27 % of the ticks. No co-infection was detected. Some samples of tick larvae showed positive results after qPCR, indicating transovarial transmission, but none of the sequences were readable.

Os micoplasmas hemotróficos (hemoplasmas) são organismos pleomórficos, epicelulares, gram negativos que infectam a superfície dos eritrócitos de diversas espécies. Devido ao fato de ser incultivável em meios de cultura tradicionais, o diagnóstico é baseado em técnicas moleculares. Em cães, a infecção pelos hemoplasmas pode causar anemia hemolítica na fase aguda, enquanto na doença crônica os sinais são inaparentes, sendo que a imunodepressão e a esplenectomia podem desencadear a doença aguda. Os objetivos do presente estudo foram avaliar a prevalência dos hemoplasmas em cães submetidos a atendimento clínico e cirúrgico no Hospital Veterinário da Universidade de Passo Fundo (HV-UPF), identificar os fatores de risco para infecçao pelos hemoplasmas e as condições clínicas na infecção natural. Para isso, foram selecionadas amostras de sangue com EDTA submetidas com propósito diagnóstico ao Laboratório de Análises Clínicas do HV-UPF. Após a verificação do controle da extração (PCR para o gene GAPDH), as amostras foram encaminhadas ao Department of Comparative Pathobiology, Purdue University (IN, USA) para as análises moleculares. Foi realizada reação em cadeia da polimerase (PCR) para os hemoplasmas felinos (‘Candidatus Mycoplasma turicencis’, ‘Candidatus Mycoplasma haemominutum’) e caninos (Mycoplasma haemocanis e ‘Candidatus Mycoplasma haematoparvum’). Ao total, foram analisadas 347 amostras, sendo que 16 foram negativas para o controle GAPDH e excluídas do estudo. A prevalência para hemoplasma foi de 6,4% (21/331) sendo 5,1% (17/331) para Mycoplasma haemocanis e 1,8% (6/331) para um organismo geneticamente semelhante ao hemoplasma felino ‘Candidatus Mycoplasma haemominutum’. Não foram encontradas amostras positivas para ‘Candidatus Mycoplasma turicencis’ e ‘Candidatus Mycoplasma haematoparvum’. Os cães positivos para hemoplasmas tinham como fatores de risco a presença de ectoparasitas (carrapatos e/ou pulgas), idade avançada e habitavam em casa. Embora tenha sido identificada a presença de correlação entre as neoplasias e feridas causadas por brigas com outros cães e a infecção pelos hemoplasmas, tais resultados foram atribuídos a uma influência da idade e a presença de ectoparasitas. Não houve variações hematológicas e bioquímicas em decorrência da infecção pelo Mycoplasma haemocanis. Refere-se, no presente estudo, o primeiro estudo relacionando a freqüência da infecção pelos hemoplasmas em cães no Brasil. / The hemotropic mycoplasmas (haemoplasmas) are pleomorphic epicellular and gramnegative organisms that do not grow in conventional culture media. The organisms infect the red blood cell surface of several species and nowadays the diagnosis is based on molecular techniques. In dogs, the acute infection causes severe hemolytic anemia, while in the chronic disease the clinical signs are unapparent. In addition splenectomy or immunosuppression may result in the acute disease. The aims of the present study were to evaluate the prevalence of haemoplasmas in dogs that were submitted to clinical or chirurgical procedures at the Veterinary Teaching Hospital of the University of Passo Fundo (UPF-HV), to identify the risk factors for infection and the clinical conditions of haemoplasma natural infection. EDTA blood samples that were submitted for diagnosis purpose at the Laboratório de Análises Clínicas of the HV-UPF were randomly selected and clinical data were obtained from the hospital database. After DNA extraction and the internal control, the samples were sent to the Department of Comparative Pathobiology at the Purdue University (IN, USA), School of Veterinary Medicine, for molecular analysis. A polymerase chain reaction (PCR) for feline haemoplasmas ('Candidatus Mycoplasma turicencis' and 'Candidatus Mycoplasma haemominutum') and canine haemoplasmas (Mycoplasma haemocanis and 'Candidatus Mycoplasma haematoparvum') was performed. A total of 347 samples were analyzed, while 16 were negative for the control GAPDH and excluded from the study. The total prevalence was 6.4% (21/331), 5.1% (17/331) for Mycoplasma Haemocanis and 1.8% (6/331) for an organism genetically similar to feline 'Candidatus Mycoplasma haemominutum' and no samples were found positive to 'Candidatus Mycoplasma turicencis' and 'Candidatus Mycoplasma haematoparvum'. The haemoplasma positive dogs lived at private homes, are old and were in contact with ectoparasites (ticks and/or fleas). Although it was identified a positive correlation between neoplasic disease, wounds caused by dog fights and the haemoplasma infection, these results were attributed to the influence of age and the vector. Hematological and biochemical variations were not identified at Mycoplasma haemocanis positive samples. This is the first study frequency of haemoplasma infection in dogs in Brazil.

Made available in DSpace on 2014-06-11T19:33:38Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-11-26Bitstream added on 2014-06-13T20:45:32Z : No. of bitstreams: 1 andrade_ag_dr_jabo.pdf: 23073379 bytes, checksum: 890e280dd903085eca5cf094bbbb6ce3 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Para o estudo da viabilidade do emprego de metodologias de sistema de informação geográfica, geoestatística e redes neurais foram desenvolvidos dois ensaios. O primeiro visou a modelagem da evolução espaço-temporal da incidência do Huanglongbing ao longo dos anos de 2004, 2007 e 2008 em cinco grupos de idade (0 a 2 anos, 3 a 5 anos, 6 a 10 anos, mais de 10 anos, todas as idades), por meio da técnica geoestatística da krigagem e da técnica de inteligência artificial de rede neurais do tipo perceptron de múltiplas camadas com topologia 3x10x10x1, treinada pelo algoritmo da propagação elástica do erro. Neste ensaio foi ainda realizada a análise do modelo do semivariograma, cálculo da média zonal dos talhões dentro de cada faixa de idade e mesorregião do estado de São Paulo, e finalmente, foi previsto o padrão espacial da incidência da doença para o ano de 2009 pelas redes neurais. O segundo ensaio consistiu em se verificar o efeito do alinhamento de plantio na severidade da mancha preta dos citros e sua implicação na qualidade do suco e produção. Neste ensaio foram selecionados talhões distribuídos ao longo do cinturão citrícola paulista com diferentes alinhamentos de plantio, onde foram analisadas as variáveis supracitadas nas linhas de plantio e nas faces expostas da planta. / For viability study of using geographic information systems, geostatistics and neural networks two experiments were carried out. The first one was focused on modeling the spatial-temporal progress of Huanglongbing incidence in 2004, 2007 and 2008 on five age groups (0 – 2 years old, 3 – 5 years old, 6 – 7 years old, more than 10 years old and all ages) using the geostatistical technique of kriguing and the artificial intelligence technique of neural networks of multi-layer perceptron type with 3x10x10x1 topology, trained by resilient error propagation. Complementing the study the analysis of semivariogram model, zonal mean calculation of blocks on each age group were realized inside the mesoregions of São Paulo state and finally the disease incidence spatial pattern for 2009 was forecasted by the neural networks. The objective of the second experiment was verifying effect of planting alignment on citrus black spot severity and its implication on juice quality and production. On this experiment was selected blocks distributed among the São Paulo state citrus belt with different planting alignments, where was analyzed those variables on the lines and exposed faces.