Made available in DSpace on 2016-01-26T18:55:43Z (GMT). No. of bitstreams: 1 Edlayne Larissa Gretter Machado Periera.pdf: 352357 bytes, checksum: a92ace5bfa90617b05e3cdaa7e4de894 (MD5) Previous issue date: 2015-03-27 / This study aimed to evaluate the limit of detection of Toxocara canis larvae in experimentally contaminated commercial bovine milk, based on a centrifuge-sedimentation technique. Firstly, bovine milk (integral and skim) samples were contaminated with 50 T. canis larvae, in order to evaluate the interference of milk fat on the recovery of the larvae. The second step of the study was to verify the influence of formalin 10% (100 µL), ether (100 µL) and combination of both solutions. The third step consisted of studying the limit of detection of larvae, using the best solution (step 2) for degreasing the milk. Samples were contaminated with aliquots of 1; 5; 10; 25 and 50 larvae. For each milk sample (1.0 mL), 15 repetitions were analyzed. It was observed that the recovery of the larvae from the skim milk was higher (p= 0.0031) than those from the integral milk. There was no significant difference (p= 0.5681) regarding the percentage of recovered larvae in comparing the degreasing solutions. Nevertheless, the formalin-ether combination was more efficient to recover the larvae (73.1%) in comparison to the ether (71.9%), the pure integral milk (70.0%) as well as the formalin (67.6%). Concerned to the limit of detection (using formalin-ether), all the samples contaminated with 5; 10; 25 and 50 larvae were positive (minimum: 62.7%). The samples contaminated with a single larva, 66.7% were positive. The technique may be useful for recovering Toxocara spp. larvae in milk samples of a wide range of animal species, either naturally or experimentally contaminated. / O objetivo do estudo foi avaliar o limiar de detecção de larvas de Toxocara canis em leite bovino comercial contaminado experimentalmente, a partir de uma técnica de centrifugo-sedimentação. Primeiramente, amostras de leite bovino comercial (integral e desnatado) foram contaminadas com 50 larvas de T. canis, para avaliação da interferência da gordura sobre a recuperação das larvas. Em uma segunda etapa, foi avaliada a ação de formalina 10% (100 µL), éter (100 µL) e combinação das soluções. A terceira etapa consistiu da verificação do limite de detecção de larvas, com uso da melhor solução desengordurante (etapa 2), em amostras de leite (1,0 mL) contendo 1; 5; 10; 25 e 50 larvas. Para cada análise de detecção do leite (1,0 mL), foram realizadas 15 repetições. Foi observado que o percentual de recuperação de larvas no leite desnatado foi significativamente maior (p= 0,0031) que o observado no leite integral. Na comparação das soluções, não houve diferença significativa (p= 0,5681) no percentual de larvas recuperadas. Entretanto, houve uma maior recuperação quando do emprego da combinação de formol-éter (73,1%) em relação ao éter (71,9%), leite integral puro (70,0%) e ao formol (67,6%). Em relação ao limite de detecção, com uso de formalina-éter, todas as alíquotas apresentaram resultado positivo (mínimo de 62,7% de larvas recuperadas), para as amostras com 5, 10, 25 e 50 larvas. Nas amostras contaminadas com uma larva, 66,7% das alíquotas foram positivas. A técnica apresentada poderá servir para recuperação de larvas de Toxocara spp. em amostras de leite de várias espécies animais, contaminados naturalmente ou experimentalmente.

Made available in DSpace on 2016-07-18T17:53:15Z (GMT). No. of bitstreams: 1 Edlayne Larissa Gretter Machado Periera.pdf: 352357 bytes, checksum: a92ace5bfa90617b05e3cdaa7e4de894 (MD5) Previous issue date: 2015-03-27 / This study aimed to evaluate the limit of detection of Toxocara canis larvae in experimentally contaminated commercial bovine milk, based on a centrifuge-sedimentation technique. Firstly, bovine milk (integral and skim) samples were contaminated with 50 T. canis larvae, in order to evaluate the interference of milk fat on the recovery of the larvae. The second step of the study was to verify the influence of formalin 10% (100 µL), ether (100 µL) and combination of both solutions. The third step consisted of studying the limit of detection of larvae, using the best solution (step 2) for degreasing the milk. Samples were contaminated with aliquots of 1; 5; 10; 25 and 50 larvae. For each milk sample (1.0 mL), 15 repetitions were analyzed. It was observed that the recovery of the larvae from the skim milk was higher (p= 0.0031) than those from the integral milk. There was no significant difference (p= 0.5681) regarding the percentage of recovered larvae in comparing the degreasing solutions. Nevertheless, the formalin-ether combination was more efficient to recover the larvae (73.1%) in comparison to the ether (71.9%), the pure integral milk (70.0%) as well as the formalin (67.6%). Concerned to the limit of detection (using formalin-ether), all the samples contaminated with 5; 10; 25 and 50 larvae were positive (minimum: 62.7%). The samples contaminated with a single larva, 66.7% were positive. The technique may be useful for recovering Toxocara spp. larvae in milk samples of a wide range of animal species, either naturally or experimentally contaminated. / O objetivo do estudo foi avaliar o limiar de detecção de larvas de Toxocara canis em leite bovino comercial contaminado experimentalmente, a partir de uma técnica de centrifugo-sedimentação. Primeiramente, amostras de leite bovino comercial (integral e desnatado) foram contaminadas com 50 larvas de T. canis, para avaliação da interferência da gordura sobre a recuperação das larvas. Em uma segunda etapa, foi avaliada a ação de formalina 10% (100 µL), éter (100 µL) e combinação das soluções. A terceira etapa consistiu da verificação do limite de detecção de larvas, com uso da melhor solução desengordurante (etapa 2), em amostras de leite (1,0 mL) contendo 1; 5; 10; 25 e 50 larvas. Para cada análise de detecção do leite (1,0 mL), foram realizadas 15 repetições. Foi observado que o percentual de recuperação de larvas no leite desnatado foi significativamente maior (p= 0,0031) que o observado no leite integral. Na comparação das soluções, não houve diferença significativa (p= 0,5681) no percentual de larvas recuperadas. Entretanto, houve uma maior recuperação quando do emprego da combinação de formol-éter (73,1%) em relação ao éter (71,9%), leite integral puro (70,0%) e ao formol (67,6%). Em relação ao limite de detecção, com uso de formalina-éter, todas as alíquotas apresentaram resultado positivo (mínimo de 62,7% de larvas recuperadas), para as amostras com 5, 10, 25 e 50 larvas. Nas amostras contaminadas com uma larva, 66,7% das alíquotas foram positivas. A técnica apresentada poderá servir para recuperação de larvas de Toxocara spp. em amostras de leite de várias espécies animais, contaminados naturalmente ou experimentalmente.

Anhydrobiotic organisms have the remarkable ability to lose extensive amounts of body water and survive in an ametabolic, suspended animation state. Distributed to various taxa of life, these organisms have evolved strategies to efficiently protect their cell membranes and proteins against extreme water loss. At the molecular level, a variety of mutually non-exclusive mechanisms have been proposed to account particularly for preserving the integrity of the cell membranes in the desiccated state. Recently, it has been shown that the dauer larva of the nematode Caenorhabditis elegans is anhydrobiotic and accumulates high amounts of trehalose during preparation for harsh desiccation (preconditioning), thereby allowing for a reversible desiccation / rehydration cycle. Here, we have used this genetic model to study the biophysical manifestations of anhydrobiosis and show that, in addition to trehalose accumulation, the dauer larvae exhibit a systemic chemical response upon preconditioning by dramatically reducing their phosphatidylcholine (PC) content. The C. elegans strain daf-2 was chosen for these studies, because it forms a constitutive dauer state under appropriate growth conditions. Using complementary approaches such as chemical analysis, time-resolved FTIR-spectroscopy, Langmuir-Blodgett monolayers, and fluorescence spectroscopy, it is shown that this chemical adaptation of the phospholipid (PL) composition has key consequences for their interaction with trehalose. Infrared-spectroscopic experiments were designed and automated to particularly address structural changes during fast hydration transients. Importantly, the coupling of headgroup hydration to acyl chain order at low humidity was found to be altered on the environmentally relevant time scale of seconds. PLs from preconditioned larvae with reduced PC content exhibit a higher trehalose affinity, a stronger hydration-induced gain in acyl chain free volume, and a wider spread of structural relaxation rates during lyotropic transitions and sub- headgroup H-bond interactions as compared to PLs from non-preconditioned larvae. The effects are related to the intrinsically different hydration properties of PC and phosphatidylethanolamine (PE) headgroups, and lead to a larger hydration-dependent rearrangement of trehalose-mediated H-bond network in PLs from preconditioned larvae. This results in a lipid compressibility modulus of ∼0.5 mN/m and 1.2 mN/m for PLs derived from preconditioned and non-preconditioned larvae, respectively. The ensemble of these changes evidences a genetically controlled chemical tuning of the native lipid composition of a true anhydrobiote to functionally interact with a ubiquitous protective disaccharide. The biological relevance of this adaptation is the preservation of plasma membrane integrity by relieving mechanical strain from desiccated trehalose- containing cells during fast rehydration. Finally, the thermo-tropic lipid phase behavior was studied by temperature-dependent ATR-FTIR and fluorescence spectroscopy of LAURDAN-labeled PLs. The results show that the adaptation to drought, which is accomplished to a significant part by the reduction of the PC content, relies on reducing thermo-tropic and enhancing lyotropic phase transitions. The data are interpreted on a molecular level emphasizing the influence of trehalose on the lipid phase transition under biologically relevant conditions by a detailed analysis of the lipid C=O H-bond environment. The salient feature of the deduced model is a dynamic interaction of trehalose at the PL headgroup region. It is proposed here that the location of trehalose is changed from a more peripheral to a more sub-headgroup-associated position. This appears to be particularly pronounced in PLs from preconditioned worms. The sugar slides deeper into the inter-headgroup space during hydration and thereby supports a quick lateral expansion such that membranes can more readily adapt to the volume changes in the swelling biological material at reduced humidity. The data show that the nature of the headgroup is crucial for its interaction with trehalose and there is no general mechanism by which the sugar affects lipidic phase transitions. The intercalation into a phosphatidylethanolamine-rich membrane appears to be unique. In this case, neither the phase transition temperature nor its width is affected by the protective sugar, whereas strong effects on these parameters were observed with other model lipids. With respect to membrane preservation, desiccation tolerance may be largely dependent on reducing phosphatidylcholine and increasing the phsophatidylethanolamine content in order to optimize trehalose headgroup interactions. As a consequence, fast mechanical adaptation of cell membranes to hydration-induced strain can be realized.

Paenibacillus larvae is a Gram-positive sporulating bacterium that causes American foulbrood (AFB). It is one of the most dangerous bacterial pathogens of the honeybee (Apis mellifera). P. larvae spores are highly infectious to bee larvae and resist physicochemical influences. P. larvae is subtyped using repPCR with ERIC primers (Enterobacterial Repetitive Integrance Consensus) into five genotypes (ERIC I-V), which possess different colony morphology, metabolism and especially virulence. There is a significant genetic variability among isolates of P. larvae, which may contribute to differences in virulence. P. larvae isolates used in this work were obtained from clinical cases of American foulbrood as well as from a debris collected from bee hives with no American foulbrood symptoms from all over the Czech Republic in cooperation with the Beekeeping Research Institute, s.r.o., Dol. The isolates were obtained from larvae and hive debris. Both virulet and avirulet strains were sequenced using the SMRT (single molecule real time) method on the Sequel platform (PacBio). This method is suitable for Whole Genome Sequencing (WGS), because it allows sequencing of long reads with high accuracy, eliminating the effect of a large number of repetitive sequences during the genome assembly. Furthermore,...

Like a large part of western Europe also from the Czech landscape is dramatically disappearing amphibians. This negative trend occurred due to several factors, some of which is undoubtedly the most significant loss of suitable breeding areas. During the years 2009 and 2010 I monitored a total of 31 tanks in a part of the former districts of Liberec, Jablonec nad Nisou and Semily, in which were multiplied frog (Rana temporaria) and toad (Bufo bufo). On these localities I've watched a few environment variables, which I assume that may affect the successful development of amphibians from egg stage to metamorphosis. The success of development, I determined by counting congeries in the spring and then finding the number of tadpoles in the tank survived the conversion. The results of multiple linear regression shows that the developmental stages of brown frog react negatively to the increasing altitude and increasing the number of eggs per unit area of the tank. On the contrary, they thrive better in larger, at least partially shaded tanks, which are subject to fluctuations in water level. For toad are suitable not very deep, partially shaded tanks, with a stable water level, vegetation and aquatic vegetation free shores. Also managed to find the eggs and tadpoles of toads react negatively to the...

Adhesive organs are widespread structures among vertebrate larvae. They allow the larvae to attach to a substrate, so that the time for the development of mouth or motoric apparatus could be prolonged. Similar structures in ascidians, larvaceans and lancelets are known too. Thus, it might be hypothesized that the presence of some type of adhesive gland could indeed represent the ancestral state for chordate larvae. Interestingly, however, whilst in most species these glands take their developmental origin in ectodermal layer, in bichir, a member of a primitive actinopterygian lineage, their origin was suggested to be endodermal already at the beginning of 20th century. Since then, however, the former study has become almost forgotten and even recent analyses do not come with new findings on this topic. Because of the essential importance of study of bichir cement glands for understanding the relationship between these structures among chordates, I have decided to focus on this subject. To obtain appropriate comparative data Xenopus, Weather loach and Ribbed newt embryos were included in this study as well. By using combination of immunohistochemical and histological techniques the endodermal origin of cement glands in bichir was proven and their morphogenesis was described into considerable details. The...

Aquatic larvae of many vertebrate lineages develop specialized, cranially located cement or attachment glands which allow them to remain attached to a substrate by means of polysaccharide secretion. The larvae can thus remain still and safe in well-oxygenated water out of reach of any predators until the digestive and locomotory apparatus fully develops. Xenopus cement gland is the most thoroughly studied example of this type of glands, since it was used as a model for the anteriormost patterning of the developing head. Based on shared expression patterns of key transcription factors and a similar ectodermal origin it has been repeatedly suggested that Xenopus cement gland is homologous to adhesive organs of teleosts and adhesive papillae of ascidians. The lack of comprehensive knowledge on this type of glands in other lineages however rendered any considerations of homology among such a distant lineages rather inconclusive. In the present work I have focused on a detailed study of the cement glands and other corresponding structures in three representatives of basal actinopterygian lineages: Senegal bichir (Polypterus senegalus), sterlet (Acipenser ruthenus), and tropical gar (Atractosteus tropicus). Using a combination of in vivo fate-mapping approaches with a Micro-CT imaging of cranial endoderm...

Some populations of newts (Taricha granulosa) possess large quantities of the neurotoxin tetrodotoxin (TTX) in their skin and eggs. Many populations of garter snake (Thamnophis sirtalis) are resistant to this toxin and can consume large numbers of newts with no negative effects. Despite the wealth of information acquired on the interaction between newts and their predator, garter snakes, very little research has been conducted on possible interactions between newts and other predators. I conducted a suite of experiments examining for the presence of other predators on newts, specifically focusing on predators of their eggs and larvae. I found a single predator, caddisfly larvae were capable of consuming the toxic eggs. Larval caddisflies are extremely abundant at one study site (775,000 caddisfly larvae per pond), and appear to be resistant to the negative effects of ingesting tetrodotoxin. After hatching, larval newts retain substantial quantities of TTX and most are unpalatable to predatory dragonfly naiads. Ovipositing female newts respond to the presence of caddisflies by depositing their eggs at the top of the water column where they are out of the reach of most predatory caddisflies. When caddisflies do consume a newt egg, some of the toxin is retained in their body tissues. Finally, caddisflies consume more newt eggs when those eggs contain less toxin versus eggs that contain large amounts of TTX. This may cause newt eggs that contain low quantities of TTX to more likely to die of predation which could ultimately drive an increase in toxicity of the adult population over time. Collectively, these findings indicate an additional player, caddisfly larvae, is a major predator of newts and could be involved in the evolution of tetrodotoxin toxicity in newts.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第19530号 / 理博第4190号 / 新制||理||1601(附属図書館) / 32566 / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 朝倉 彰講師宮崎 勝己, 教授 曽田 貞滋 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

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