Orientadores: Urara Kawazoe, Elizabeth Santin / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-26T10:12:03Z (GMT). No. of bitstreams: 1 Iuspa_MariaAparecidaMelo_M.pdf: 891258 bytes, checksum: 6e794e24b82f1c94278fc775155f9813 (MD5) Previous issue date: 2014 / Resumo: Foi realizado experimento em frangos de corte machos de duas linhagens, com duração de 42 dias. Foram utilizados 800 frangos de corte machos de um dia de idade alojados em um galpão. O delineamento experimental foi realizado em blocos, num fatorial 2 x 2 (duas linhagens infectadas e não infectadas). Cada tratamento foi replicado oito vezes. Os tratamentos experimentais foram T1, aves da linhagem Cobb 500; T2, aves da linhagem Ross 308, ambos inoculados via oral, aos 14 dias de idade em dose única de 1 x 106 oocistos; T3, aves da linhagem Cobb 500; e T4, aves da linhagem Ross 308, não inoculados. Os critérios de avaliação mensurados foram peso médio das aves, consumo de ração, conversão alimentar ajustada, eficiência alimentar, ganho de peso, escore de lesão, contagem de oocistos e análise de células caliciformes. O escore de lesão das duas linhagens infectadas foi avaliado aos seis dias após a inoculação. O número de oocistos médio por grama de excretas foi obtido entre o quarto e 10º dias após a inoculação. Os resultados de desempenho não mostraram diferenças estatísticas significativas entre as duas linhagens infectadas e as linhagens não infectadas para todos os períodos avaliados, com exceção do peso médio, aos 21 dias de idade. A linhagem Cobb apresentou um peso médio superior ao da linhagem Ross (p<0,001) aos 21 dias de idade. O escore de lesão médio da linhagem Ross foi maior que o da linhagem Cobb (p=0,006). O mesmo foi verificado para o número de oocistos médio por grama de excretas, para o qual a linhagem Ross apresentou maior contagem do que a linhagem Cobb (p<0,0001). Não houve diferença significativa na contagem de células caliciformes de duodeno e jejuno entre os tratamentos. A diferença de desempenho e resistência entre as linhagens infectadas observadas através do peso médio aos 21 dias de idade (sete dias após a inoculação), escore de lesão e quantidade de oocistos por grama de excretas desapareceu conforme o crescimento dos animais até o final do experimento. As reduções relativas de peso médio foram menores na linhagem Cobb, quando comparado à linhagem Ross, e decresceram conforme os animais chegavam à idade final do experimento nas duas linhagens (42 dias). Os aumentos relativos de conversão alimentar foram maiores para a linhagem Cobb quando comparada a linhagem Ross e também decresceram conforme os animais chegavam à idade final do experimento nas duas linhagens (42 dias). A utilização de linhagens de curva de crescimento rápido e curva de crescimento lenta de acordo com o objetivo produtivo (abate precoce ¿ frangos "griller" e abate tardio ¿ frangos pesados) deve ser considerada uma vez que as respostas de desempenho e resistência ao desafio de E. acervulina destas linhagens são distintos, podendo extrair máximo benefício da resistência genética conforme a idade de abate e desafio / Abstract: Evaluation of susceptibility and resistance between two strains of male chicken inoculated with Eimeria acervulina was carried out during 42 days. A total of 800 male broiler chickens with one day of age was used and placed in a floor-pen unit within a typical broiler house. The experimental design was in a randomized block designed in a 2 x 2 factorial (two strains infected and not infected) with eight replications. The treatments were T1, Cobb 500 broiler chicken strain; T2, Ross 308 broiler chicken strain, both were orally inoculated at 14 days of age, with a single dosage of 1 x 106 oocysts; T3, Cobb 500 broiler chicken strain; and T4, Ross 308 broiler chicken, not inoculated. The following criteria were used: average weight, feed consumption, adjustment of feed conversion, feed efficiency, weight gain, lesion score, oocysts counting, caliciform cells analysis. The results of performance did not show statistical differences between infected strains and between non infected strains during all periods, with exception to the average weight of 21 days of age. The Cobb strain showed a higher average weight compared with Ross strain (p<0.001) at 21 days of age. Lesion score evaluated at 6 days after inoculation was higher for Ross strain compared with the Cobb strain (p=0.006). The same result was observed for oocysts counting. The Ross strain presented a higher number of oocysts than the Cobb strain (p<0.0001). There was no statistical difference between strains in caliciform cells of duodenum and jejunum in all treatments. The results showed performance and susceptibility differences between infected strains on the average weight at 21 days of age (seven days after inoculation), lesion score and number of oocysts per gram of excreta; this difference disappeared according the birds were growing up, until the end of trial. The relative average of the weight reduction of the Cobb strain was lower than the Ross, and the relative average weight reduction in both strains decreased according birds were growing up, until the end of trial (42 days of age).The relative feed conversion of the Cobb strain was higher than the Ross strain and the relative feed conversion in both strains decreased according birds were growing up until the end of trial (42 days of age). The use of strains of fast growth and slow growth curve according to the production goal (early-slaughter chickens "griller" and heavy chicken) should be considered once the responses of performance and resistance to E. acervulina challenge of these strains are distinct and it is possible to have the maximum benefit from genetic resistance according to slaughter age and challenge / Mestrado / Relações Antrópicas, Meio Ambiente e Parasitologia / Mestra em Biologia Animal

Orientador: Valquíria Cação Cruz-Polycarpo / Resumo: O objetivo deste estudo foi avaliar o efeito do butirato de sódio microencapsulado (BSM) em substituição ao antibiótico sobre o desempenho, hematologia, peso de órgãos do TGI, escores de lesão intestinal e contagem de oocistos nas excretas de frangos de corte desafiados com Eimeria spp. Para isso foram utilizados 1.050 pintos machos Ross, distribuídos em DIC, com seis tratamentos: CN (controle negativo)- ração basal (RB) (aves não desafiadas); CND- RB (aves desafiadas); 0,10%- RB + 1.000 mg/kg de BSM (aves desafiadas); 0,15%- RB + 1.500 mg/kg de BSM (aves desafiadas); 0,20%- RB + 2.000 mg/kg de BSM (aves desafiadas); AVL- RB + avilamicina (aves desafiadas), com cinco repetições. Aos 16 dias de idade, as aves foram inoculadas oralmente e individualmente com oocistos de Eimerias e as aves de CN, inoculadas com solução salina, para que também sofressem o estresse da inoculação. Observou-se maiores GPM e CRM nas aves não desafiadas em comparação às desafiadas na fase de 1 a 21 d, e na fase final de criação e melhor CA e FEP, mostrando o poder da inoculação com o protozoário. Não houve diferença nas variáveis hematológicas e bioquímicas entre as diferentes dietas fornecidas, porém frangos submetidos às Eimerias apresentaram queda nos valores normais após desafio, e demonstraram recuperação ao final da criação. Na alometria de órgãos do TGI, o peso do fígado apresentou-se superior nas aves que receberam aditivo. Já, o peso do pâncreas, mostrou-se superior nas aves suplementadas com... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The objective of this study was to evaluate the effect of microencapsulated sodium butyrate (MSB) in substitution to antibiotics on performance, hematology, weight of organs of the GIT, intestinal injury scores and oocyst count in the excreta of broilers challenged with Eimeria spp. For this, 1,050 male Ross chicks were used, distributed in completely randomized design, with six treatments: NC - basal diet (BD) (non-challenged birds); NCC- BD (challenged birds); 0.10% - BD+1,000 mg/kg of MSB (challenged birds); 0.15% - BD+1,500 mg/kg of MSB (challenged birds); 0.20% - BD+2,000 mg/kg of MSB (challenged birds); AVL - BD + avilamycin (challenged birds), with five repetitions. At 16 days of age, the birds were inoculated orally and individually with Eimerias oocysts and the NC birds were inoculated with saline solution, so that they also suffered the stress of inoculation. Higher ABW and AFI were observed in the unchallenged birds compared to those challenged in the 1 to 21 day phase, and in the final breeding phase, better FCR and PEF, showing the power of inoculation with the protozoan. There was no difference in hematological and biochemical variables between the different diets provided, but chickens subjected to inoculation with Eimerias showed a drop in normal values after challenge, and showed recovery at the end of the rearing period. In the allometry of organs of the TGI, the liver weight was higher in the birds that received additive. The weight of the pancreas, on the ... (Complete abstract click electronic access below) / Mestre

The in vitro production of gametocytes and oocysts of the apicomplexan parasite genus Eimeria is still a challenge in coccidiosis research. Until today, an in vitro development of gametocytes or oocysts had only been shown in some Eimeria species. For several mammalian Eimeria species, partial developments could be achieved in different cell types, but a development up to gametocytes or oocysts is still lacking. This study compares several permanent cell lines with primary fetal cells of the black rat (Rattus norvegicus) concerning the qualitative in vitro development of the rat parasite Eimeria nieschulzi. With the help of transgenic parasites, the developmental progress was documented. The selected Eimeria nieschulzi strain constitutively expresses the yellow fluorescent protein and a macrogamont specific upregulated red tandem dimer tomato. In the majority of all investigated host cells the development stopped at the second merozoite stage. In a mixed culture of cells derived from inner fetal organs the development of schizont generations I-IV, macrogamonts, and oocysts were observed in crypt-like organoid structures. Microgamonts and microgametes could not be observed and oocysts did not sporulate under air supply. By immunohistology, we could confirm that wild-type E. nieschulzi stages can be found in the crypts of the small intestine. The results of this study may be helpful for characterization of native host cells and for development of an in vitro cultivation system for Eimeria species.

A coccidiose aviária é uma doença entérica causada por protozoários parasitas do gênero Eimeria. Visando uma maior compreensão dos mecanismos moleculares envolvidos na regulação do ciclo de vida dos parasitas, foram geradas 15.000 seqüências expressas (ORESTES) para cada uma das três espécies mais importantes: E. tenella, E. maxima e E. acervulina. O presente trabalho consistiu no desenvolvimento de componentes de anotação automática de seqüências para o sistema EGene, plataforma previamente desenvolvida pelo nosso grupo (Durham et al. Bioinformatics 21: 2812-2813, 2005) para a construção de processamentos encadeados (pipelines). Estes componentes foram utilizados para a construção de pipelines de anotação automática de seqüências-consenso obtidas a partir da montagem dos ORESTES de Eimeria spp. A anotação consistiu na identificação dos genes e atribuição da função dos respectivos produtos protéicos, baseando-se em um conjunto de evidências. As seqüências também foram classificadas e quantificadas utilizando-se um vocabulário controlado de termos de ontologia gênica (GO). / Avian coccidiosis is an enteric disease caused by protozoan parasites of the genus Eimeria. Aiming at obtaining a better understanding of the molecular mechanisms that regulate the life cycle of the parasites, our group generated 15,000 expressed sequences (ORESTES) for each one of the three most important species: E. tenella, E. maxima and E. acervulina. In the present work, we report the development of a set of components for the automated sequence annotation through EGene, a platform for pipeline construction previously described by our group (Durham et al. Bioinformatics 21: 2812-2813, 2005). These components were used to construct pipelines for the automated annotation of assembled sequences of ORESTES of Eimeria spp. The annotation process consisted in the identification of genes and the corresponding protein function based on a set of evidences. The sequences were also mapped and quantified using a controlled vocabulary of gene ontology (GO) terms.

O gênero Eimeria compreende um grupo de protozoários da classe Coccidia que infecta uma grande variedade de hospedeiros. Um total de sete espécies distintas Eimeria podem infectar a galinha doméstica causando enterites com graves prejuízos econômicos. A identificação das espécies pode ser feita através da análise microscópica das diferentes características morfológicas dos oocistos, um dos estágios de desenvolvimento do parasita. Alternativamente, ensaios moleculares baseados na amplificação de alvos específicos de DNA também podem ser utilizados. Em ambos os casos, requer-se um laboratório especializado e, principalmente, pessoal altamente treinado. Neste trabalho é relatada uma abordagem computacional para a extração automática de características para a representação da forma das distintas espécies de Eimeria. Foram utilizadas imagens digitais do protozoário nas quais aplicou-se técnicas de processamento de imagens e visão computacional para sua representação morfológica, formando três grupos de características: medidas geométricas, caracterização da curvatura, e quantificação da estrutura interna. A morfologia dos protozoários foi representada por um vetor de características constituído por 14 dimensões, o qual constituiu o padrão de entrada para o processo de classificação. Para o reconhecimento dos padrões, foram usados dois classificadores Bayesianos, utilizando-se como funções de verossimilhança a Gaussiana e a de Dirichlet, respectivamente. O primeiro classificador apresentou as melhores taxas de acerto, enquanto o segundo demonstrou melhor desempenho segundo a análise por curvas ROC. Como prova de princípio de que o sistema poderia ser utilizado por usuários leigos para o diagnóstico à distância de parasitas, foi implementado o COCCIMORPH, um sistema de diagnóstico de Eimeria em tempo real. O sistema permite o envio de imagens via web, assim como o seu pré-processamento e classificação remotos, obtendo-se o resultado do diagnóstico em tempo real. Essa abordagem totalmente integrada e implementada é inédita para o diagnóstico de parasitas. Entre suas vantagens principais está o fato de que o diagnóstico pode ser obtido sem a necessidade do transporte físico de amostras biológicas para um laboratório de referência, evitando assim riscos de contaminação do ambiente. Para o treinamento do sistema, foram obtidas centenas de micrografias de cada uma das sete espécies de Eimeria que infectam a galinha doméstica. Essas imagens também foram usadas para a construção de um banco de acesso público de imagens (The Eimeria Image Database). Além disso, a metodologia de diagnóstico foi também aplicada e testada com onze espécies Eimeria de coelho doméstico. Com isso, foram gerados dados inéditos de morfometria, micrografias adicionais para o banco de imagens, e um sistema de classificação para esse conjunto adicional de parasitas. Finalmente, foram determinadas as distâncias entre as diferentes espécies de Eimeria, calculadas a partir dos dados morfométricos. As árvores de distância revelaram uma topologia muito similar com árvores obtidas a partir da inferência filogenética usando-se marcadores moleculares como o gene 18S de rRNA ou genomas mitocondriais. / The Eimeria genus comprises a group of protozoan parasites that infect a wide range of hosts. A total of seven different Eimeria species infect the domestic fowl, causing enteritis with severe economical losses. Species identification can be performed through microscopic analysis of the distinct morphological characteristics of the oocysts, a developmental stage of the parasite. Alternatively, molecular assays based on the amplification of specific DNA targets can also be used. In both cases, a well equipped laboratory and, especially, highly qualified personnel are required. In this work, we report a computational approach for the automatic feature extraction for shape representation of the different Eimeria species. Digital images of the parasites were used in order to apply image processing and computational vision techniques for shape characterization. Three groups of morphological features were constituted: geometric measures, curvature characterization, and internal structure quantification. The protozoan morphology was represented by a 14-dimension feature vector, which was used as the input pattern for the classification process. Two Bayesian classifiers were used for pattern recognition, using as a likelihood function the normal and the Dirichlet, respectively. The former classifier presented the best correct classification rates, whereas the latter showed a better performance in ROC curve analyses. As a proof of principle that this system could be utilized by end-users for a long-distance parasite diagnosis, we implemented COCCIMORPH, an integrated system for the real-time diagnosis of Eimeria spp. The system presents an interface for image uploading. Image preprocessing and diagnosis are performed remotely and the results displayed in real-time. This fully integrated and implemented system constitutes a novel approach for parasite diagnosis. Among the several advantages of the system, it is noteworthy that no biological sample transportation is required between the farm and the reference laboratory, thus avoiding potential environment contamination risks. To train the system, we used hundreds of micrographs of each one of the seven Eimeria species of domestic fowl. These images were used to compose a public image repository (The Eimeria Image Database). In addition, our diagnosis methodology was extended to the eleven Eimeria species that infect the domestic rabbit. With this integrated approach, a totally novel set of images and morphometric data of rabbit Eimeria were incorporated to the image database and, also to the remote diagnosis system. Finally, distance trees of the distinct Eimeria species of domestic fowl were computed from the morphometric data. The trees revealed a very similar topology with trees obtained with molecular phylogenetic markers such as the 18S rRNA gene and mitochondrial genomes.

Das Überleben und die Vermehrung der parasitär lebenden Apicomplexa setzen eine effiziente Synthese von Phospholipiden während ihres gesamten Lebenszyklus voraus. In dieser Arbeit nutzten wir zunächst Eimeria falciformis um den Prozess der Lipid-Biogenese in Sporozoiten zu untersuchen. Durch Lipidomics-Analysen wurde das Auftreten von zwei exklusiven Lipiden, Phosphatidylthreonin (PtdThr) und Inositolphosphorylceramid. Der Parasit exprimiert fast das gesamte Lipid-Biogenese- Netzwerk aus eukaryotischen und prokaryotischen Enzymen. Toxoplasma gondii diente als genmanipulierbarer Ersatz für die Untersuchung der Eimeria-Enzyme, mit dem wir ein stark räumlich segmentiertes Netzwerk der Lipidsynthese im Apicoplast, ER, Golgi und Mitochondrium zeigen konnten. Ebenso legte die Komplementierung einer T. gondii-Mutante mit einer PtdThr-Synthase von E. falciformis eine konvergente Funktion von PtdThr für den lytischen Zyklus von Kokzidien-Parasiten nahe. Außerdem setzten wir T. gondii als etablierten Modelorganismus ein, um die De- novo-Synthese und die metabolische Rolle eines bedeutenden Lipidvorläufers, CDP- Diacylglycerin (CDP-DAG), zu untersuchen. Wir konnten zwei phylogenetisch divergente CDP-DAG-Synthase (CDS) Enzyme in T. gondii nachweisen. Das eukaryotisch-typische TgCDS1 und das prokaryotisch-typische TgCDS2 lokalisieren im ER bzw. im Apicoplast. Der konditionierte Knockdown von TgCDS1 bremst das Parasitenwachstum stark ab, was den fast vollständigen Verlust der Virulenz im Mausmodell hervorruft. Das restliche marginale Wachstum der TgCDS1 Mutante wird durch zusätzliche Deletion der TgCDS2 verhindert. Lipidomics-Analysen zeigten eine signifikante und spezifische Abnahme der Phosphatidylinositol (PtdIns)- und Phosphatidylglycerol (PtdGro)-Level bei Verlust der TgCDS1- bzw. TgCDS2-Gene. Zusammengenommen zeigt unsere Arbeit ein Phospholipid-Biogenese-Modell mit erstaunlicher Kooperation verschiedener Organellen und einem extensiven Lipidtransport im Parasiten. / The survival and proliferation of apicomplexan parasites oblige efficient synthesis of phospholipids throughout their life cycles. Here, we first deployed Eimeria falciformis to investigate the process of lipid biogenesis in sporozoites. Lipidomics analyses demonstrate the occurrence of two exclusive lipids phosphatidylthreonine (PtdThr) and inositol phosphorylceramide along with other prototypical lipids. The parasite expresses nearly the entire lipid biogenesis network, which is an evolutionary mosaic of eukaryotic- and prokaryotic-type enzymes. Using Toxoplasma gondii as a gene- tractable surrogate to examine the Eimeria enzymes, we show a highly compartmentalized network of lipid synthesis distributed primarily in the apicoplast, ER, Golgi and mitochondrion. Likewise, trans-species complementation of a T. gondii mutant with a PtdThr synthase from E. falciformis suggests a convergent function of PtdThr in promoting the lytic cycle in coccidian parasites. We also employed the well-established model parasite T. gondii to explore de novo synthesis and metabolic roles of one major lipid precursor CDP-diacylglycerol (CDP- DAG). We report the occurrence of two phylogenetically divergent CDP-DAG synthase (CDS) enzymes in T. gondii. Eukaryotic-type TgCDS1 and prokaryotic-type TgCDS2 reside in the ER and apicoplast, respectively. Conditional knockdown of TgCDS1 severely attenuates parasite growth, which translates into a nearly complete loss of virulence in a mouse model. Residual growth of the TgCDS1 mutant is abolished by subsequent deletion of TgCDS2. Lipidomics analyses reveal significant and specific decline in phosphatidylinositol (PtdIns) and phosphatidylglycerol (PtdGro) upon loss of TgCDS1 and TgCDS2, respectively. Taken together, our work establishes a phospholipid biogenesis model involving significant inter-organelle cooperation and lipid trafficking in apicomplexan parasites.

In den vorliegenden Studien wurden verschiedene diagnostische Verfahren zum Nachweis von Protozoen und Nematoden im Hinblick auf Sensitivität, Arbeitsaufwand und Kosten miteinander verglichen. Zudem wurde die Eignung der PCR zur molekularen Charakterisierung der Cryptosporidium spp. exemplarisch an Igelkotproben getestet. Bei der Untersuchung von 90 Ferkelkotproben auf I. suis war die Sensitivität eines Kotausstriches mit nachfolgender Autofluoreszenzmikroskopie (AM) signifikant höher als bei einem Flotationsverfahren (FV) mit NaCl-Zucker-Lösung und bei dem kombinierten Sedimentations-Flotations-Verfahren (KSFV) mit verschiedenen Flotationslösungen (NaCl, ZnSO4, NaCl-Zucker-Lösung) mit nachfolgender Lichtmikroskopie. Zudem ist der Arbeitsaufwand für die AM deutlich geringer als bei dem FV und KSFV. Die höheren apparativen Kosten für die AM sind bei hohem Probendurchsatz durch den geringeren Zeitaufwand und der höheren Sensitivität gerechtfertigt. Die Anzahl Kryptosporidien-positiver Proben war bei der Untersuchung von 103 Kälberkotproben auf Cryptosporidium sp. mittels Enzymimmunoassays (EIA; ProSpecT® Cryptosporidium Microplate Assay) im Vergleich zur Karbolfuchsin-Färbung (CF) nach HEINE (1981) und der modifizierten-Ziehl-Neelsen-Färbung (MZN) nach HENRIKSEN u. POHLENZ (1982) am höchsten und signifikant höher als bei der Anwendung der MZN, wenn 10 Blickfelder durchmustert wurden. Bei der Untersuchung von 74 Igelkotproben auf Cryptosporidium sp. mittels EIA (ProSpecT®), einem immunochromatographischen Verfahren (FASTest® CRYPTO Strip), der MZN nach HENRIKSEN u. POHLENZ (1981) und einem direkten Immunfluoreszenz-Test (IFA; MERIFLUOR Cryptosporidium/Giardia) wurden in 9 (EIA), 10 (FASTest®), 11 (MZN) und 12 (IFA) Proben Cryptosporidium sp. nachgewiesen. Der Arbeitsaufwand des FASTest® und der CF ist mit dem EIA vergleichbar, während der IFA und die MZN mehr Zeit benötigen. Die Anwendung des FASTest®, des IFA und des EIA ist mit höheren Kosten verbunden als bei den Färbemethoden, können aber gut in den Arbeitsablauf eines diagnostischen Labors eingefügt werden und sind einfach auszuwerten. Darüber hinaus wurden 45 Kotproben, welche bis zu 27 Tage bei verschiedenen Temperaturen (+6 °C, +16 °C, +30 °C, +40 °C) gelagert wurden, untersucht, um einen Einfluss der Temperatur auf das Untersuchungsergebnis von EIA, CF und MZN zu ermitteln. Während sich die Anzahl positiver Proben bei der Untersuchung mit den Färbemethoden temperatur- und zeitabhängig reduzierte, wurde das Untersuchungsergebnis mittels EIA von der Lagerungstemperatur nicht beeinflusst, so dass ungekühlt transportierte Proben vorzugsweise mit dem EIA untersucht werden sollten. Dagegen ist die CF aufgrund ihrer einfachen und preiswerten Durchführung zur Untersuchung einer hohen Anzahl an Proben geeignet, sofern eine ununterbrochene Kühlung der Proben gewährleistet ist und diese innerhalb von drei Tagen untersucht werden. Der FASTest® ist zur Anwendung in Tierarztpraxen und Ställen geeignet, da zur Untersuchung kein Mikroskop benötigt wird und die Resultate schnell vorliegen. Die Verwendung des IFA, der Kryptosporidien-Oozysten und Giardien-Zysten nachweist, bietet sich vor allem bei Proben an, die auf beide Protozoen untersucht werden sollen. Das Vorkommen der Kryptosporidiose bei unterentwickelten und geschwächten Igeln, welche zum Überwintern in Igelstationen aufgenommen werden, ist hoch. Von 188 untersuchten Igelkotproben konnten in 29,8 % der Proben Cryptosporidium spp. nachgewiesen werden. Durch die Genotypisierung der Kryptosporidien aus 15 positiven Igelkotproben mittels RFLP-PCR basierend auf dem 18S rRNA-Gen konnte in allen untersuchten Proben die Präsenz von C. parvum gezeigt werden. Mit Hilfe der Multilocus-Sequenz-Typisierung der Fragmente des 60kDa Glycoprotein-Gens, des 18S rRNA-Gens, des Actin-Gens und des 70 kDa Hitzeschockprotein-Gens konnten drei verschiedene Subtypen-Familien (IIa, IIc und eine neue als VIIa vorgeschlagene Subtypen-Familie) erkannt werden. Die von den Igeln ausgeschiedenen Kryptosporidien-Oozysten mit zum Teil nachgewiesenem zoonotischen Potential (IIa Subtypen-Familie) könnten eine Infektionsquelle für den Menschen sein, aber auch ein antropozoonotisches Potential (IIc Subtypen-Familie) sollte in Betracht gezogen werden, so dass die Hygiene in den Igelstationen einen hohen Stellenwert einnehmen sollte. Die Untersuchungsergebnisse zum Nachweis von Eimeria-Arten beim Kalb von 70 Sammelkotproben, hergestellt aus 10 Einzelkotproben (SKP10), bzw. von 30 Sammelkotproben, zusammengesetzt aus 5 Einzelkotproben (SKP5), wurden mit denen der zugehörigen Einzelkotproben (EKP) verglichen. Die Resultate der EKP (arithmetischer Mittelwert) und der zugehörigen SKP weisen mit den signifikant häufigeren Abweichungen im Bereich von bis zu 100 Oozysten pro Gramm Kot (OpG) eine geringe Differenz zwischen den beiden Verfahren auf. Durch den sicheren Nachweis von Eimeria-Oozysten bei einem erwarteten Oozystengehalt von nur 202 OpG (SKP10) und 122 OpG (SKP5) ist die Untersuchung von Kälbersammelkotproben, eine Methode mit geringem Arbeitsaufwand und geringen Untersuchungskosten, zum Nachweis einer klinischen oder subklinischen Kokzidiose geeignet. Bei 51 Pferdekotproben wurde jeweils dreimal das kombinierte Sedimentations-Flotations-Verfahren (KSFV), wobei die Entnahme von verschiedenen Lokalisationen der Kotprobe (aus der Randregion, dem Inneren oder aus beiden Lokalisationen) erfolgte, und jeweils dreimal das KSFV mit vorheriger Homogenisierung einer größeren Kotmenge zum Nachweis von Nematodeneier durchgeführt. Eine Anhäufung der Strongyliden- und Ascarideneier in einem bestimmten Bereich der Proben konnte durch die Untersuchungen der verschiedenen Lokalisationen (á 10 g Kot) nicht nachgewiesen werden, so dass eine weitgehend homogene Verteilung dieser Nematodeneier in einer Pferdekotprobe wahrscheinlich ist. Zudem konnten die Untersuchungsergebnisse des KSFV, bei welchem 10 g Kot untersucht werden, durch die vorherige Homogenisierung einer größeren Probenmenge nicht verbessert werden. Zum Nachweis von Nematoden beim Pferd sollte dem Labor eine ausreichende Probenmenge (ca. 50 g) zugesandt werden. Die Homogenisierung einer größeren Probenmenge vor der Durchführung einer diagnostischen Methode, bei der Aliquote von mindestens 10 g Kot Verwendung finden, ist unnötig. / The studies presented were carried out to compare different diagnostic methods for detection of protozoa and nematodes regarding sensitivity, expenditure of human labour and costs. Besides, the ability of the PCR for the molecular characterization of the Cryptosporidium spp. was tested exemplarily in faecal samples of hegdehogs. The examination of ninety faecal samples of suckling piglets showed a significantly higher sensitivity of faecal smears examined by autofluorescence microscopy (AM) compared to the flotation method (FV) using NaCl-sucrose solution and the combined sedimentation-flotation method (KSFV) using different flotation solutions (NaCl, ZnSO4, NaCl-sucrose) scanned by bright field microscopy. Moreover the expenditure of human labour by AM is considerably lower than FV and KSFV. The costs related to equipment for AM is justified in case of high sample throughput and by superior sensitivity. The enzyme immunoassay (EIA; ProSpecT® Cryptosporidium Microplate Assay) was the most sensitive method for diagnosis of cryptosporidiosis in calves (n = 103) compared to the carbol fuchsin (CF; HEINE 1981) and modified Ziehl-Neelsen (MZN; HENRIKSEN a. POHLENZ 1982) staining techniques. The sensitivity of the EIA was significantly higher than the MZN, if ten fields of view were scanned. 74 faecal samples of hedgehogs were examined with the EIA (ProSpecT®), an immunochromatographic method (FASTest® CRYPTO Strip), the MZN (HENRIKSEN u. POHLENZ (1981)) and a direct immunofluorescent assay (IFA; MERIFLUOR Cryptosporidium/Giardia). Cryptosporidium sp. were detected in 9 (EIA), 10 (FASTest®), 11 (MZN) und 12 (IFA) faecal samples. The hands on time of the FASTest® and CF is comparable to EIA while the IFA and MZN are more time-consuming. The examination of the FASTest®, IFA and EIA is combined with higher costs than the staining techniques, but they can be integrated in the work flow of a routine diagnostic laboratory easily and evaluation is simple. Moreover 45 faecal samples stored up to 27 days at different temperature (+6 °C, +16 °C, +30 °C, +40 °C) were examined to evaluate the influence of temperature on the results of EIA, CF and MZN. While the number of the positive samples of stained smears decreased in a temperature and time-dependent manner, the results of the EIA were not influenced by sample storage at any temperature, so that samples transported without cooling should be examined preferably by EIA. Nevertheless the CF due to its simplicity and low costs is suited for scanning of a high number of samples, if they were cooled continuously and examined within three days. The FASTest® is qualified for use in veterinary practice and stables, because the examination requires no microscope and the results are obtained immediately. The IFA, which can detect Crypotsporidium oocysts as well as Giardia cysts, is suited especially for faecal samples suspected to contain both protozoa. Cryptosporidial infections are very frequent in hedgehogs which are admitted for hibernation to hedgehog rehabilitation centres because of their insufficient body weight and weakness. Cryptosporidium spp. were detected in 29.8 % of 188 faecal samples of hedgehogs. The genotyping of Cryptosporidium spp. by PCR and RFLP-PCR based on the 18S ribosomal RNA gene were performed on 15 faecal samples of hedgehogs positive for Cryptosporidium spp. and suggested the presence of C. parvum in all samples. Multilocus sequence typing on partial 60 kDa glycoprotein gene, 18S rRNA gene, actine gene, 70 kDa heat shock protein gene sequences revealed 3 different subtype families: IIa, IIc and a new proposed as VIIa subtype family. Some of the Cryptosporidium oocysts excreted from hedgehogs are zoonotical (IIa subtype family) or anthropozoonotic(IIc subtype family). Thus hygienic measurements to avoid transmission are essential in hedgehog rehabilitation centres. The results of examination of 70 pooled faecal samples originating from 10 calves (SKP10) and 30 pooled faecal samples originating from 5 calves (SKP5) for detection of Eimeria spp. were compared with the arithmetic means of opg (oocysts per gram of faeces) counts of the respective single 10 or 5 samples. A low difference between both methods of less than 100 opg was significantly more frequently observed than higher differences. Low values of 202 opg and 122 opg were reliably detected in SKP10 und SKP5, respectively, and thus examination of pooled faecal samples appears to be suitably sensitive and cost effective to detect clinical and subclinical coccidiosis in calves. 51 faecal samples of horses were examined three times by KSFV for nematode eggs by taking aliquots from different locations of the same faecal samples (from the margin, from inside and from both locations). Thereafter the KSFV with the homogenisation of a larger amount of faeces was also carried out three times. The examination of samples from the different locations (each 10 g of faeces) delivered no evidence for accumulation of nematode eggs (strongyles and Parascaris equorum) in the faeces and thus the distribution of the nematode eggs appears sufficiently homogeneous in faecal samples of horses. Homogenisation of a larger amount of faeces did not improve the results of coproscopy. For diagnostic purposes 50 g faeces per sample should be shipped to the laboratory. The homogenisation of a larger amount of faeces before using a diagnostic method is dispensable, if aliquots of 10 g faeces are examined.

In der vorliegenden Arbeit wurden die Ergebnisse von 263 Einzeltierverfolgungen in 12 Betrieben in verschiedenen Regionen in Deutschland, Belgien, Frankreich und der Tschechischen Republik zur Stallkokzidiose der Kälber zusammengefasst ausgewertet. Während diesen Untersuchungen wurden 5840 Kotproben beurteilt. Dabei wurden das Auftreten und die Ausprägung der Eimeriose der Kälber analysiert und potentielle Einflussfaktoren untersucht und ein verlässlicher Schwellenwert für die Bewertung der Oozystenausscheidung ermittelt. Weiterhin wurden in 16 Betrieben bei 23 gemeinsam aufgestallten Kälbergruppen Spezies-Prävalenzuntersuchungen über einen Zeitraum von fünf Wochen durchgeführt. Hierfür wurden 5133 Proben ausgewertet, davon 3519 mit Teil- und 1614 mit vollständiger Differenzierung. In allen Betrieben traten die Kotkzidiosen als eine Mischinfektion verschiedener Eimeria-Arten auf. Dabei herrschte in der Regel in jedem Betrieb eine der beiden pathogensten Spezies vor. Diese Prädominanz blieb auch über mehrere Jahre gleich in den Betrieben. Die Herkunft der Kälber hatte einen Einfluss auf den Infektionsverlauf. In Betrieben mit ausschließlich eigener Nachzucht verläuft die Kokzidiose als eingipflige Infektion, bei Zukaufbetrieben ist der Infektionsverlauf zweigipflig. Ein signifikant erhöhtes Risiko des Auftretens einer klinisch ausgeprägten Kokzidiose bei Aufstallung auf Stroheinstreu im Vergleich zur Haltung auf Spaltenboden konnte bewiesen werden (p = 0,005). In der Verfolgungsuntersuchung korrelierten die Kokonsistenzwerte mit den nachgewiesenen OpG in den Kotproben positiv signifikant (0,135 Korrelationskoeffizient; p = 0,000). Für das Auftreten von Durchfall konnte eine statistisch bewiesene lineare Korrelation mit der Oozystenausscheidung für diverse Schwellenwerte festgestellt werden (p = 0,000). Bei einem Grenzwert von 500 OpG lag der Korrelationskoeffizient bei 0,149. Die Korrelationswerte erhöhten sich nicht wesentlich bei Festlegung eines höheren Grenzwertes für die Oozystenausscheidung oder sanken sogar (0,153 bis 0,121). Bei der Verrechnung von Durchfallvorkommen mit gleichzeitiger, potentiell relevanter Oozystenausscheidung mit den verschiedenen Schwellenwerten der Oozystenausscheidung konnte der höchste Zusammenhang zwischen dem Durchfallgeschehen und dem Schwellenwert von 500 OpG bewiesen werden (0,633 Korrelationskoeffizient; p = 0,000). Daher kann ein Wert von 500 OpG pathogener Eimeria spp. als geeigneter Schwellenwert für die Feststellung einer relevanten Oozystenausscheidung angesehen werden. Bei gleichzeitigem Auftreten von Durchfall ist von einer maßgeblichen Beteiligung der Eimerien auszugehen. Ein gehäuftes Auftreten von mit Oozystenausscheidung assoziierten Durchfällen trat bei Tieren ohne oder ohne potentiell relevante Oozystenausscheidung (‚rK -’) signifikant seltener auf (p = 0,000) als bei Tieren mit mindestens 500 aufsummierten OpG während des gesamten Beobachtungszeitraumes (‚rK +’). Kälber der Kategorie ‚Kok-Kat 1’ hatten signifikant niedrigere Kotkonsistenzwerte und weniger Durchfälle als Tiere der Subpopulation ‚rK -’ (p = 0,000). Dagegen hatten die Tiere der Auswertungsgruppe ‚Kok-Kat 2’ statistisch bewiesen in allen Durchfall-Kategorien höhere Werte bzw. ein häufigeres Durchfallvorkommen als beide anderen Subpopulationen (p = 0,000 für alle Vergleiche). Wässrige Durchfälle mit Beimengungen traten, mit Ausnahme einer Einzelbeobachtung in Gruppe ‚rK -’, ausschließlich bei Kälbern der Auswertungsgruppe ‚Kok-Kat 2’ auf. Sowohl Kälber der Gruppe ‚rK +’ als auch ‚Kok-Kat 2’-Tiere (jeweils p = 0,000) und Kälber mit potentiell relevanter Oozystenausscheidung, aber ohne gleichzeitiges Durchfallgeschehen, (‚Kok-Kat 1’) (p = 0,005) hatten signifikant geringere relative Gewichtszunahmen als Tiere ohne bzw. ohne potentiell relevante Oozystenausscheidung (‚rK -’). Eine lineare Korrelation der Ausscheidung der pathogenen Spezies E. bovis und E. zuernii mit den absoluten (-0,098 Korrelationskoeffizient; p = 0,005) und relativen Gewichtszunahmen (-0,170 Korrelationskoeffizient; p = 0,000) konnte statistisch bewiesen werden. Bei Haltung auf Stroheinstreu zeigten Tiere ohne bzw. mit weniger als 500 ausgeschiedenen OpG im Untersuchungszeitraum (‚rK -’) signifikant höhere relative Zunahmen im Vergleich zu Tieren mit potentiell relevanter Oozystenausscheidung (p = 0,000). Dabei war es ohne Bedeutung, ob diese Kälber ein gleichzeitiges Durchfallgeschehen zeigten oder nicht. Besonders zum Tragen kommen diese Unterschiede in der Gewichtsentwicklung in den Wochen mit den höchsten Oozystenausscheidungen bei zweigipfligem Infektionsverlauf. Unter den Milchviehkälbern nahmen Tiere ohne relevante Oozystenausscheidung signifikant mehr relatives Gewicht zu als Kälber mit kumulativ mindestens 500 OpG im Untersuchungszeitraum (p = 0,004). Dies galt sowohl für Tiere mit gleichzeitigem Durchfall ‚Kok-Kat 2’ (p = 0,002) als auch tendenziell für Kälber der Gruppe ‚Kok-Kat 1’ (p = 0,059). Mastviehkälber der Gruppe ‚rK -’ zeigten signifikant höhere relative Zunahmen im Vergleich zu Tieren mit potentiell relevanter Oozystenausscheidung ‚rK +’ (p = 0,039). Dies galt auch in Relation zu den Kälbern der Auswertungsgruppe ‚Kok-Kat 1’ (p = 0,029). Während der Prävalenzuntersuchungen wurden insgesamt neun verschiedene Eimeria-Arten nachgewiesen. In Einzelkotproben wurden zwischen einer und neun verschiedene Spezies beobachtet. Unter den in Europa als heimisch bekannten Arten wurden während dieser Untersuchung lediglich E. wyomingensis, E. brasiliensis und E. bukidnonensis nicht gefunden. E. ellipsoidalis hatte sowohl die höchste Inzidenz (20,99 %) als auch die größte Intensität (arithmetischer Mittelwert von 765963,37 OpG), gefolgt von E. bovis und E. zuernii. Zudem wurde in Aufzuchtbetrieben E. ellipsoidalis in der Regel als erste Eimeria-Spezies, gefolgt von E. auburnensis und den pathogenen Arten E. zuernii und E. bovis, nachgewiesen. Die seltensten Spezies waren E. canadensis und E. pellita. Eimeria pellita wurde als letzte Art erst ab der fünften Woche nach Aufstallung beobachtet. E. cylindrica trat vermehrt in den Betrieben in Belgien und Frankreich auf. Diese Art sowie E. canadensis wurden nur in Betrieben in Bayern, Belgien und Frankreich festgestellt. E. pellita hatte, neben E. canadensis, die geringsten Prävalenzen, Nachweise wurden vor allem für zentral gelegene Betriebe sowie im Süden des Untersuchungsgebietes dokumentiert. Die Übereinstimmung der gefundenen Varianzen der Speziesprävalenzen der vorliegenden Untersuchung mit den Daten epidemiologischer Studien in den verschiedenen Regionen aus der Literatur bestätigt einen repräsentativen Charakter der Untersuchungsbetriebe. Dies belegt zusätzlich die Allgemeingültigkeit der festgestellten Einflussfaktoren auf das Auftreten und die Auswirkungen der Stallkokzidiose der Kälber. / A total number of 263 calves housed on 12 different farms in several regions in Germany, Belgium, France and the Czech Republic were followed individually in tracking studies and data was compiled and analysed to investigate factors influencing occurrence and severity of bovine eimeriosis of housed calves. The same data was used for development and verification of a suitable threshold indicating relevant oocyst excretion. Within the tracking studies a total number of 5840 faecal samples were examined for faecal consistency, oocyst counts of pathogenic E. bovis and E. zuernii excretion carried out and individual body weight development was documented regularly. Additionally 23 groups of animals on 16 farms were observed for a period of five weeks and 5133 faecal samples examined for oocyst excretion and Eimeria species differentiated. All study sites were positive for mixed coccidia species infections. Nevertheless all farms except one showed a predominance of one pathogenic Eimeria species, which remained unchanged in different groups of animals and even in different years of investigations. Animal origin, i.e. groups of animals representing own breeding or originating of only one source in contrast to groups of calves coming from several origins, influences the course of the coccidiosis infection. Farms with only one single and permanent animal origin or raising exclusively the own breeding show coccidiosis with a single peak of oocyst excretion. On farms housing groups of animals of various origins the course of infection and oocyst shedding has typically two peaks with an interval of two to three weeks. The risk for development of clinical coccidiosis rises significantly if animals were housed on straw bedding compared to slatted-floor (p = 0.005). Faecal scores correlated significantly (p = 0.000) with the intensity of oocyst excretion with a positive correlation coefficient of 0.135. For occurrence of diarrhoea a positive linear correlation with the oocyst excretion was statistically proved (p = 0.000) for various thresholds. At a threshold of 500 opg of E. bovis and E. zuernii the correlation coefficient rised to 0.149 and correlation coefficients did not rise distinctly or even got down if higher thresholds were used (values between 0.153 and 0.121). Focusing only on potentially coccidiosis related diarrhoea the threshold of 500 opg of E. bovis and E. zuernii proved to result in the highest correlation (0.633; p = 0.000) of all tested threshold levels. Therefore the threshold of 500 opg of E. bovis and E. zuernii can be accounted modest and reliable to detect a relevant oocyst excretion in individual faecal samples as well as in compiled samples. In cases of coincidental diarrhoea coccidiosis can be considered as a major factor. Increased numbers of days with diarrhoea in coincidence with an oocyst excretion (‘Kokass-DF’) within the observation period were seen significantly more often (p = 0.000) in animals with a potentially relevant oocyst excretion (‘rK +’) of at least 500 summed up opg of E. bovis and E. zuernii in comparison to calves without such an oocyst excretion (‘rK -‘). Significantly lower faecal scores and fewer days with diarrhoea were documented for calves of the group ‘Kok-Kat 1’ in contrast to animals of group ‘rK -‘ (p = 0.000). Nevertheless significantly higher faecal scores and more days with diarrhoea than both other groups were calculated for those calves meeting the inclusion criteria for group ‘Kok-Kat 2’ (p = 0.000 for all comparisons). Additionally liquid faeces or faeces with constituencies were seen only in this group, except for one single sample of a calf of group ‘rK -‘. Calves of evaluation group ‘rK +’ as well as both subpopulations representing group ‘Kok-Kat 2’ and calves with potentially relevant oocyst excretion but without diarrhoea associated to an oocyst excretion (‘Kok-Kat 1’) showed significant lower values for relative body weight increases in comparison to animals without relevant oocyst excretion throughout the complete study period of five weeks (‘rK -‘) (p = 0.000 vs. ‘rK +’ and vs. ‘Kok-Kat 2‘; p = 0.005 compared with ‘Kok-Kat 1’). A negative linear correlation between oocyst excretion of pathogenic Eimeria spp. and absolute (-0.098 correlation coefficient; p = 0.005) as well as relative body weight gain (-0.170 correlation coefficient) was verified statistically (p = 0.000). Animals housed on straw bedding and belonging to the group ‘rK -‘ gained relatively more body weight in comparison to calves housed in the same stables and meeting the inclusion criterium of group ‘rK +’, i.e. excreting at least 500 summed up opg of pathogenic E. spp., (p = 0.000) within the total study period. The presence of coincidental diarrhoea had no impact on impaired body weight development of animals with a potentially relevant coccidia excretion. Differences in body weight development were most distinct within the weeks of highest intensities in oocyst excretion according to a course of infection with two peaks. Within the subpopulation of dairy calves those animals belonging to evaluation group ‘rK -‘ developed significantly higher relative body weight gains compared to group ‘rK +’ (p = 0.004). Similar results were found for animals of group ‘Kok-Kat 2’ (p = 0.002) and a statistical tendency was calculated for group ‘Kok-Kat 1’ (p = 0.059) in comparison to group ‘rK -‘. Analogous to the differences in dairy calves animals on fattening farms without relevant oocyst excretion (‘rK -’) had significantly higher relative body weight gains compared to calves of evaluation group ‘rK +’ (p = 0.039) and animals of group ‘Kok-Kat 1’ (p = 0.029) of the same breeds and farms. Nine different Eimeria spp. were detected during the prevalence studies. In single individual samples a minimum of one and up to nine different species were found. Twelve Eimeria spp. are known to be endemic in Europe of which only E. brasiliensis, E. bukidnonensis and E. wyomingensis were not present in any faecal sample in this study. E. bovis and E. zuernii were only second to E. ellipsoidalis which had the highest prevalence (20.99 %) as well as the highest intensity (765963.37 mean opg) in the faecal samples examined. In breeding farms E. ellipsoidalis was the first species to be found in faecal examination in most cases, followed by E. auburnensis and the pathogenic species E. zuernii and E. bovis. E. canadensis and E. pellita were detected only in a low number of samples. E. pellita was observed for the first time at the faecal samplings five weeks after stabling and mainly in farms situated in the central and southern region of the prevalence study. E. canadensis and E. cylindrica were most prominent in farms situated in Belgium and France. Variances in prevalence of the species observed are in conformity with those to be found in recent literature according to the different regions of Europe. This may indicate a representative character of the farms participating in this study and therefore universal validity of the results and impactfactors on coccidiosis in calves described in this manuscript.

http://www.marth.com/tkuehn/diss/diss_32_abstract.html / http://www.marth.com/tkuehn/diss/diss_32_abstract.html

The genus Eimeria (Apicomplexa, Coccidia) provides a wide range of different species with different hosts to study common and variable features within the genus and its species. A common characteristic of all known Eimeria species is the oocyst, the infectious stage where its life cycle starts and ends. In our study, we utilized Eimeria nieschulzi as a model organism. This rat-specific parasite has complex oocyst morphology and can be transfected and even cultivated in vitro up to the oocyst stage. We wanted to elucidate how the known oocyst wall-forming proteins are preserved in this rodent Eimeria species compared to other Eimeria. In newly obtained genomics data, we were able to identify different gametocyte genes that are orthologous to already known gam genes involved in the oocyst wall formation of avian Eimeria species. These genes appeared putatively as single exon genes, but cDNA analysis showed alternative splicing events in the transcripts. The analysis of the translated sequence revealed different conserved motifs but also dissimilar regions in GAM proteins, as well as polymorphic regions. The occurrence of an underrepresented gam56 gene version suggests the existence of a second distinct E. nieschulzi genotype within the E. nieschulzi Landers isolate that we maintain.