Master of Science / Department of Agronomy / William T. Schapaugh / Remote sensing technologies might serve as indirect selection tools to improve phenotyping to differentiate genotypes for yield in soybean breeding program as well as the assessment of soybean cyst nematode (SCN), Heterodera glycines. The objective of these studies were to: i) investigate potential use of spectral reflectance indices (SRIs) and canopy temperature (CT) as screening tools for soybean grain yield in an elite, segregating population; ii) determine the most appropriate growth stage(s) to measure SRI’s for predicting grain yield; and iii) estimate SCN population density among and within soybean cultivars utilizing canopy spectral reflectance and canopy temperature. Experiment 1 was conducted at four environments (three irrigated and one rain-fed) in Manhattan, KS in 2012 and 2013. Each environment evaluated 48 F4- derived lines. In experiment 2, two SCN resistant cultivars and two susceptible cultivars were grown in three SCN infested field in Northeast KS, in 2012 and 2013. Initial (Pi) and final SCN soil population (Pf) densities were obtained. Analyses of covariance (ANCOVA) revealed that the green normalized vegetation index (GNDVI) was the best predictive index for yield compared to other SRI’s and differentiated genotype performance across a range of reproductive growth stages. CT did not differentiate genotypes across environments. In experiment 2, relationships between GNDVI, reflectance at single wavelengths (675 and 810 nm) and CT with Pf were not consistent across cultivars or environments. Sudden death syndrome (SDS) may have confounded the relationships between remote sensing data and Pf. Therefore, it would be difficult to assess SCN populations using remote sensing based on these results.

Master of Science / Department of Computing and Information Sciences / Mitchell L. Neilsen / This research is a part of BREAD PHENO, a PhenoApps BREAD project at K-State which combines contemporary advances in image processing and machine vision to deliver transformative mobile applications through established breeder networks. In this platform, novel image analysis segmentation algorithms are being developed to model and extract plant phenotypes. As a part of this research, the traditional Watershed segmentation algorithm has been extended and the primary goal is to accurately count and characterize the seeds in an image. The new approach can be used to characterize a wide variety of crops. Further, this algorithm is migrated into Android making use of the Android APIs and the first ever user-friendly Android application implementing the extended Watershed algorithm has been developed for Mobile field-based high-throughput phenotyping (HTP).

The 129S2 inbred mouse strain is often used as a background strain in the production of genetically altered mice (i.e. knockout and transgenic mice). It is important to establish the behavioral phenotype of wild-type mice before making comparisons to genetically altered mice. Also, those comparisons can assist in the evaluation and interpretation of the in vivo effects of drugs. The drug discrimination assay measures the subjective effects of drugs and provides a measure of underlying neuropharmacological mechanisms responsible for the discriminative stimulus properties of drugs. The present study established the atypical antipsychotic drug clozapine as a discriminative stimulus in male 129S2 inbred mice and compared clozapine’s discriminative stimulus properties in 129S2 mice to C57BL/6 and DBA/2 inbred mice. By comparing the discriminative stimulus properties between inbred strains of mice we hope to obtain a fuller picture of the underlying neuropharmacological mechanisms of antipsychotic drugs.

Schizophrenia is a debilitating mental disorder that causes a large economic burden and is prevalent across all cultures and countries around the world. Although both environmental factors and genetics are known to play an important role in the etiology of schizophrenia, the exact role of genetics and its interaction with environmental factors in an individual’s predisposition to develop schizophrenia is poorly understood. Schizophrenia is characterized by symptoms that include positive symptoms (e.g. delusions, hallucinations, disorganized thinking and speech), negative symptoms (e.g. avolition, anhedonia, depressive-like behavior), and cognitive dysfunctions (e.g. executive functioning deficits in learning and memory, attention, and vigilance). Genomic screening has identified polymorphisms of the vesicular monoamine transporter 1 (VMAT1) gene (SLC18A1) that are associated with schizophrenia and bipolar disorder. The current study represents the first extensive phenotyping of both young and aged mice in which the VMAT1 gene (SLC18A1) has been deleted. The results demonstrated behavioral effects of deleting the VMAT1 gene that may relate to aspects of schizophrenic-like behavioral changes in this model. Specifically, young VMAT1 knockout mice displayed significant deficits in sensorimotor gating in the prepulse inhibition (PPI) task and in the acquisition of operant learning in the autoshaping task. When exposed to a mild stressor (24 hours of food deprivation), young VMAT1 knockout mice displayed a significant reduction in locomotor activity that was not evident under free-feeding conditions. Thus, young VMAT1 knockout mice showed deficits in tasks that model positive symptoms and cognitive deficits seen in schizophrenia; however, they did not display differences in behaviors related to models of the negative symptoms of schizophrenia or deficits in tasks designed to measure motor skills. While less extensive phenotyping was conducted in aged VMAT1 knockout mice, there were no significant deficits evident in any of the assays conducted in older animals. These findings demonstrated that deletion of the VMAT1 gene has behavioral effects that appear to be mediated by changes in brain monoamine function and changes in response to stressors (i.e. food deprivation) that may reflect changes in adrenal gland monoamine function.

L'un des défis auxquels sont confrontés les producteurs de fruits est de réussir à maintenir d'année en année un équilibre entre croissance végétative et production. La productivité des arbres fruitiers au cours des années est étroitement liée à leur développement. Intégrer les caractères architecturaux dans les programmes de sélection pourrait donc, améliorer la régularité de production et aider à optimiser la gestion des cultures. Dans cette thèse, nous avons étudié le déterminisme génétique des caractères architecturaux chez l'olivier (Olea europaea L. subsp. Europaea), en incluant le développement végétatif et reproducteur. L'olivier a un grand intérêt en raison de l'importance de l'huile d'olive et des olives dans l'alimentation humaine. L'étude a porté sur une descendance issue du croisement entre les variétés 'Olivière' et ‘Arbequina' qui a été cultivée en verger dans deux environnements contrastés. En premier lieu, nous avons étudié le déterminisme génétique de la croissance et ramification des arbres durant la phase juvénile sur un site en considérant trois échelles d'observation: arbre, unité de croissance et entrenœuds. Les interactions entre les facteurs liés à l'ontogénie de l'arbre ainsi que les facteurs génotype et environnement ont été prises en compte. Des modèles génétiques, incluant les effets année de croissance et/ou ordre de ramification, environnement, génotype et leurs interactions respectives, ont été construits en intégrant une fonction de variance et une structure de covariance lorsque cela était nécessaire. Après une étape de sélection de modèle, les facteurs impactant significativement l'architecture de l'arbre ont été identifiés et les valeurs d'héritabilité au sens large ont été estimées. Ces résultats nous ont permis de définir à quel moment au cours de l'ontogenèse de l'olivier et à quelle échelle d'observation, les caractères de croissance et ramification sont déterminés génétiquement. De plus, cette étude a permis d'investiguer la plasticité des caractères architecturaux et leur stabilité entre environnements contrastés. Enfin, les changements associés à l'acquisition des facultés reproductives ont été observés. Quand tous les descendants ont atteint l'âge adulte, j'ai étudié les bases génétiques du développement reproducteur. La stratégie était basée sur (i) une décomposition d'un sous échantillon d'unités de croissance localisées à la périphérie de la couronne de l'arbre en variables quantitatives liées à la fois aux processus de floraison et fructification en relation avec leur croissance et ramification (ii) une évaluation annuelle du rendement des arbres durant quatre années. L'observation d'arbres 'ON' ou ‘OFF' pour une année donnée a révélé des tendances de production régulière vs irrégulière au sein de la descendance. Après avoir développé une nouvelle carte génétique, une recherche QTLs associés aux caractères reproductifs a été réalisée. Des QTLs présentant les effets des deux parents et des co-localisations ont été identifiés. Cette étude a mis en évidence le schéma de développement de l'olivier pendant les phases juvénile et mature mettant en évidence l'existence de gradients ontogéniques se traduisant par des caractères héritables qu'à la périphérie de l'arbre. Une stratégie de phénotypage adaptée aux caractéristiques architecturales de l'olivier a été proposée. Enfin, les descendants montrant une supériorité intéressante par rapport à leurs parents ont été identifiés. Ces génotypes pourraient être valorisés dans les programmes de sélection futurs pour la création de variétés innovantes. / One of the most challenging questions that fruit growers are facing is to maintain trees in a stable balance between production and vegetative growth from year to year. Fruit trees productivity over years is closely linked to their development. Integrating architectural traits in breeding programs could thus, optimise cultivation management and improve bearing regularity. Here, we investigate the genetic determinism of architectural traits in the olive tree (Olea europaea L. subsp. europaea) including vegetative and reproductive behaviour. The olive tree raises high interest because of the importance of olive oil and olives in the human diet. The segregating population under study derived from a cross between ‘Olivière' and ‘Arbequina' cultivars and was grown on two orchards under contrasting environments. First, we investigated the genetic determinism of juvenile growth and branching traits in one site at three different scales: whole tree, growth unit and internodes. Interaction between tree ontogeny, genetic and environmental factors were considered. Genetic models, including the year of growth, environment, genotype effects and their interactions, were built with variance function and covariance structure of residuals when necessary. After a model selection step, morphogenetic factors impacting significantly tree architecture were identified and heritabilities were estimated. These finding allow us to define when during the olive tree ontogeny and at which observation scale, growth and branching traits are under genetic influence. Moreover, progenies plastic response to contrasted environments was highlighted and traits showing significant genotype-by-environment effect were identified. Changes over time associated to the acquisition of reproductive competence were further observed. Once all progenies attempt adulthood, we studied the genetic basis of reproductive behaviour. Our strategy was based on (i) a decomposition of adult growth units at the crown periphery in quantitative variables related to both flowering and fruiting process in relation to their growth and branching (ii) an annual assessment of individual trees yield. The observation of ‘on' or ‘off' olive trees in a given year over four years revealed patterns of regular vs. irregular bearing. After developing a new genetic map, a QTL mapping was carried out on reproductive traits, leading to the identification of QTLs with effects from both parents and co-localizations. This study gives an overview of olive tree development during juvenility and maturity periods showing the existence of ontogenic trends, which result in traits heritable mostly at the tree periphery. A phenotyping strategy adapted to its architectural characteristics is proposed. Finally, progenies showing interesting superiority in comparison to their parents were identified and could constitute interesting sources for innovative materials in future selection programs.

The main objective of the present thesis was to propose a procedure to optimize genotypic information value in small dairy cattle populations and investigate the impacts of including genotypes and phenotypes of cows chosen by different strategies over the performance of genome-wide association studies and genomic selection. The first study was designed to propose innovative methods that could support alternative inference over population structure in livestock populations using graph theory. It reviews general aspects of graphs and how each element relates to theoretical and practical concepts of traditional pedigree structure studies. This chapter also presents a computational application (PedWorks) built in Python 2.7 programming language. It demonstrates that graph theory is a suitable framework for modeling pedigree data. The second study was aimed asses how graph community detection algorithms could help unraveling population partition. This new concept was considered to develop a method for stablishing new cow genotyping strategies (community-based). Results obtained showed that accounting for population structure using community detection for choosing cows to get included in the reference population may improve results from genomic selection. Methods presented are easily applied to animal breeding programs. The third study aimed to observe the impacts of different genotyping strategies (including the proposed community-based) over the ability to detect quantitative trait loci in genome-wide association studies. Distinct models for genomic analysis were also tested. Results obtained showed that including cows with extreme phenotypic observations proportionally sampled from communities can improve the ability to detect quantitative trait loci in genomic evaluations. The last chapter was designed study possible deleterious impacts of the presence of preferential treatment (in different levels) in a small dairy cattle population environment over accuracy and bias of genomic selection. Different proportions of cows with artificially increased phenotypic observations were included in the reference population. Observed results suggest that both accuracy and bias are affected by the presence of preferential treatment of cows in the evaluated population. Preferential treatment is expected to have much more effect on the performance of genomic selection in small than in large dairy cattle populations for the higher (proportional) value of the information from cows in such reduced-size breeds. / O principal objetivo da presente tese foi propor um procedimento capaz de otimizar o valor da informação genotípica em pequenas populações de gado de leite e investigar os impactos da inclusão de genótipos e fenótipos de vacas escolhidas por diferentes estratégias sobre o desempenho de estudos de associação genômica ampla e seleção genômica. O primeiro estudo foi delineado para elaborar um método que permita uma inferência alternativa sobre a estrutura populacional de populações de animais de produção usando como base a teoria de grafos. Este revê os aspectos gerais de grafos e como cada elemento se relaciona com conceitos teóricos e práticos de estudos de estrutura de pedigree tradicionais. Este capítulo também apresenta um aplicativo computacional (PedWorks) construído em linguagem de programação Python 2.7. Resultados observados demonstraram que a teoria de grafos é uma estrutura adequada para modelar dados de pedigree. O segundo estudo teve como objetivo avaliar como os algoritmos de detecção de comunidades de grafos poderiam ajudar revelar o particionamento de uma população. Este novo conceito foi considerado para desenvolver um método para o estabelecimento de novas estratégias de genotipagem de vacas (baseadas em comunidades). Os resultados obtidos mostraram que a contabilização da estrutura populacional usando a detecção de comunidades para a escolha de vacas a serem incluídas na população de referência pode melhorar os resultados da seleção genômica. Os métodos apresentados sugerem ser facilmente introduzidos em programas de melhoramento animal. O terceiro estudo teve como objetivo observar os impactos de diferentes estratégias de genotipagem (incluindo a anteriormente proposta baseada em comunidades) sobre a capacidade de detectar locos relacionados características quantitativas por meio de estudos de associação genômica ampla. Modelos distintos para análise genômica também foram testados. Os resultados obtidos mostraram que incluir vacas com observações fenotípicas extremas amostradas proporcionalmente das comunidades pode melhorar a capacidade de detectar locos de características quantitativas em avaliações genômicas. O último capítulo foi desenhado para estudar possíveis impactos deletérios da presença de tratamento preferencial no ambiente de pequenas populações de gado leiteiro sobre resultados da seleção genômica. Diferentes proporções de vacas com observações fenotípicas aumentadas artificialmente foram incluídas na população de referência. Os resultados observados sugerem que tanto a acurácia quanto o viés são afetados pela presença de tratamento preferencial de vacas na população avaliada. Espera-se que o tratamento preferencial tenha muito mais efeito sobre o desempenho da seleção genômica em populações pequenas de gado de leite que em grandes populações devido a maior relevância das informações de vacas em raças de tamanho reduzido.

A mudança anual de penas é um componente crítico do ciclo biológico de todas as espécies de aves e pode modificar as respostas imunológicas individuais, sendo que suas consequências são pouco estudadas. Neste contexto, uma vez que são escassos os estudos acerca do perfil clínico e parâmetros imunológicos de pinguins-de-Magalhães em diferentes fases da muda, este estudo propôs avaliar determinados aspectos relacionados a esses perfis, em animais mantidos em ambiente controlado. Foram estudados 21 indivíduos adultos (dez machos e 11 fêmeas) e os parâmetros avaliados foram: teste de hipersensibilidade cutânea tardia à fitohemaglutinina (FHA) com a avaliação histopatológica de biópsias que incluíram a análise qualitativa e quantitativa dos tipos celulares presentes na reação e no controle às 6, 24 e 48 horas; parâmetros clínicos (massa corpórea e hematologia); fagocitose e burst oxidativo; fenotipagem e resposta linfoproliferativa. O teste de FHA demonstrou diferença significativa na espessura da membrana interdigital de ambas as patas inoculadas com FHA e com o controle (PBS) quando comparadas antes da inoculação e antes da biópsia às 24 e 48 horas. A análise histomorfométrica revelou não haver diferenças significativas na frequência relativa de granulócitos (heterófilos/ eosinófilos), linfócitos, macrófagos ou trombócitos entre as patas inoculadas com FHA e PBS em nenhum dos momentos analisados. Este resultado sugere que a diferença na espessura da membrana interdigital das patas inoculadas seja em decorrência do edema e não do infiltrado inflamatório. Para analisar os efeitos do ciclo da muda nos parâmetros clínicos e hematológicos, os animais foram divididos em três grupos: pré-muda (-5 a 0 dias da muda), muda (10º ao 21º dia da muda) e padrão basal (>120 dias da muda). Os resultados mostraram que em relação ao momento basal: houve aumento significativo na massa corpórea dos animais no momento da pré-muda; diminuição significativa de proteínas totais, hematócrito, eritrócitos totais e leucócitos totais na pré-muda e muda. Não houve diferença no número total de monócitos e heterófilos, contudo, houve eosinofilia e linfopenia na pré-muda e muda, respectivamente. Com relação aos parâmetros imunológicos, houve aumento no burst oxidativo gerado pelo estímulo biológico (Staphylococcus aureus) na pré-muda em relação à muda e ao padrão basal; ocorreu aumento do burst oxidativo gerado pelo estímulo químico (éster de forbol) na pré-muda em relação à muda. Em relação à fagocitose, houve diminuição desta capacidade dos leucócitos dos animais na pré-muda e muda em relação ao padrão basal quando o agente foi bacteriano; entretanto, o mesmo não foi observado para a fagocitose de leveduras (Zymosan A); houve diminuição de linfócitos T CD4+ circulantes na pré-muda e muda em relação ao padrão basal e o índice de proliferação de linfócitos aumentou durante o período de muda em relação ao basal. Esses resultados tomados em conjunto sugerem que as mudanças fisiológicas observadas durante o ciclo da muda interferem na modulação de parâmetros imunológicos e são sugestivas de serem consequência do prejuízo energético sofrido, mesmo em animais mantidos em ambientes controlados / The annual replacement of feathers is a critical component of the biological cycle of all bird species. Molt can modify their individual immune response, and the consequences of this are not well known. Within this context, and the few studies on the clinical profile and immunological parameters of Magellanic penguins at different stages of their molt, this study aimed to evaluate certain aspects related to the clinical profiles of animals kept in a controlled environment. A total of 21 adult individuals (ten males and 11 females) was analyzed and the parameters evaluated in this study were: delayed-type hypersensitivity test to phytohemagglutinin (PHA) based on the histopathological evaluation of foot web biopsies that included qualitative and quantitative analysis of cell types present in the essay and control at 6, 24 and 48 hours; clinical parameters (body mass and hematology); phagocytosis and oxidative burst; phenotyping and lymphoproliferative response. The PHA test showed significant differences in the thickness of the webbing of both feet inoculated with PHA and control (PBS), when compared before inoculation and before biopsy at 24 and 48 hours. Histomorphometric analysis revealed no significant differences in the relative frequencies of granulocytes (heterophils/ eosinophils), lymphocytes, macrophages, thrombocytes, or between the feet inoculated with PHA and with PBS in any of the samples. This result suggests that the difference in thickness of the foot webbing is due to edema and not an inflammatory infiltrate. To analyze the effects of the molting cycle on clinical and hematological parameters, the birds were divided into three groups: pre-molt (from -5 days to the start of the molt, day 0), molt (10th to 21st days of molt) and baseline (>120 days after the completion of the molt). The results showed that in comparison with baseline values: a significant increase in body mass occurred during pre-molt; and a significant decrease in total protein, hematocrit, total erythrocytes and total leukocytes occurred during pre-molt and molt. There was no difference in the total numbers of monocytes and heterophils, however, there were eosinophilia and lymphopenia during pre-molt and molt, respectively. There was an increase in the oxidative burst generated by the biological stimulus (Staphylococcus aureus) in the pre-molt compared to molt and baseline levels; and there was an increased oxidative burst generated by the chemical stimuli (phorbol esters) in the pre-molt in relation to the molt. There was a decreased capacity of phagocytosis for the leukocytes in animals during pre-molt and molt compared to baseline when the agent was bacterial; however, this was not observed for the phagocytosis of yeast (Zymosan A). There was a decrease of circulating CD4+ T lymphocytes in the pre-molt and molt compared to baseline, and the lymphocyte proliferation index increased during the molt compared to baseline. The results suggest that the physiological changes observed during the molt cycle does interfere with the modulation of immune parameters, and are suggestive of resulting as a consequence of from the high energy demands of molt, even for birds kept in controlled environments

Phenotypic data is of crucial importance for plant breeding in estimating a plant's biomass. Traits such as leaf area and plant height are known to be correlated with biomass. Image analysis and computer vision methods can automate data analysis for high-throughput phenotyping. Many methods have been proposed for plant phenotyping in controlled environments such as greenhouses. In this thesis, we present multiple methods to estimate traits of the plant crop sorghum from images acquired from UAV and field-based sensors. We describe machine learning techniques to extract the plots of a crop field, a method for leaf counting from low-resolution images, and a statistical model that uses prior information about the field structure to estimate the center of each plant. We also develop a new loss function to train Convolutional Neural Networks (CNNs) to count and locate objects of any type and use it to estimate plant centers. Our methods are evaluated with ground truth of sorghum fields and publicly available datasets and are shown to outperform the state of the art in generic object detection and domain-specific tasks. <br><br>This thesis also examines the use of crowdsourcing information in video analytics. The large number of cameras deployed for public safety surveillance systems requires intelligent processing capable of automatically analyzing video in real time. We incorporate crowdsourcing in an online basis to improve a crowdflow estimation method. We present various approaches to characterize this uncertainty and to aggregate crowdsourcing results. Our techniques are evaluated using publicly available datasets.<br>

Os testes pré-transfusionais são o conjunto de técnicas imuno-hematológicas aplicadas nas amostras do receptor e do doador com a finalidade de fornecer uma unidade de hemácias compatível e segura para a transfusão. Além da fenotipagem ABO e RhD obrigatória nos testes pré-transfusionais, a determinação do perfil antigênico de outros antígenos eritrocitários de importância clínica, melhora a segurança transfusional, especialmente para pacientes previamente aloimunizados ou candidatos à transfusões crônicas. Para tais pacientes, conhecer o fenótipo eritrocitário permite a seleção de concentrados de hemácias com o mesmo fenótipo, o que previne reações transfusionais hemolíticas, assim como nova aloimunização. Indivíduos que apresentam as hemácias sensibilizadas \"in vivo\" por anticorpos anti-eritrocitários, como ocorre na anemia hemolítica autoimune (AHAI), terão o teste direto da antiglobulina (TDA) positivo, dificultando a realização da fenotipagem. O TDA positivo provoca resultados falsos positivos especialmente quando são utilizados para a fenotipagem soros comerciais da classe IgG que exigem a fase de antiglobulina humana, ou teste indireto da antiglobulina (TIA), para leitura. Em 1982 Edwards, Moulds e Judd propuseram a utilização da técnica da cloroquina para dissociar os complexos de antígenoanticorpo preservando a membrana eritrocitária e permitindo a fenotipagem. Atualmente é a técnica mais utilizada nos centros brasileiros. A técnica da glicina-ácida/EDTA foi primeiramente descrita por Louie, Jieng e Zeroulis em 1986, porém é pouco utilizada por não haver os reagentes prontos comercialmente no mercado brasileiro. O presente estudo avaliou a técnica da glicina-ácida-EDTA com reagentes preparados \"in house\" em paralelo à técnica da cloroquina, que é atualmente a técnica aplicada no Hemocentro de Ribeirão Preto. Para isso, amostras de hemácias de doadores (n=50) com fenótipo conhecido foram sensibilizadas com anticorpos humanos de especificidade conhecida e ambas as técnicas foram aplicadas. Foi verificado ainda o efeito da técnica sobre a expressão dos antígenos eritrocitários (Fya, Fyb, Jka, Jkb, S e s) através da fenotipagem das amostras que tiveram o TDA negativado. A viabiliadade dos anticorpos presentes no eluato recuperado após tratamento com a glicina- ácida/EDTA também foi testada. A glicina-ácida/EDTA foi efetiva em negativar o TDA em 33 amostras (66%), comparável com a cloroquina que negativou 28 amostras (56%). O eluato apresentou-se viável no tratamento com a glicina-ácida/EDTA e não foi constatada destruição dos antígenos eritrocitários após o tratamento. Concluímos que a técnica da glicina- ácida/EDTA pode ser utilizada como uma opção no tratamento de hemácias com TDA positivo com a vantagem de seu tempo de execução ser inferior ao da cloroquina (2 minutos x 30 a 120 minutos), o que lhe torna útil em situações de maior urgência. Quando ambas as técnicas são utilizadas em hemácias diferentes de uma mesma amostra a efetividade está significativamente aumentada (p<0,05). / The pre-transfusion tests are the group of immunohematology techniques applied in the receptor and donor samples in order to provide a unit of red blood cells compatible and safe for transfusion. In addition to the obligatory ABO and RhD phenotyping in the pretransfusion tests, the determination of the antigenic profile of other red cells antigens of clinical importance improves transfusion safety, especially for patients previously alloimmunized or candidates for chronic transfusion. For such patients, knowing the phenotype allows the selection of red blood cell concentrates with the same phenotype, which prevents hemolytic transfusion reactions, as well as new alloimmunization. Individuals who have antibody-coated red blood cells sensitized in vivo, such as in autoimmune hemolytic anemia (AHAI), will have a direct antiglobulin test (DAT) positive, making it difficult to perform phenotyping. Positive DAT causes false positive results especially when commercially available IgG serums which require the human antiglobulin phase or indirect antiglobulin test (IAT) for reading are used for phenotyping. In 1982 Edwards, Moul ds and Judd proposed the use of the chloroquine technique to dissociate antigen-antibody complexes by preserving the erythrocyte membrane and allowing phenotyping. It is currently the most used technique in Brazilian centers. The EDTA/glycine-acid technique was first described by Louie, Jieng and Zeroulis in 1986, but it is little used because commercially available reagents are not available in the Brazilian market. The present study evaluated the EDTA/glycine-acid technique with reagents prepared in house in parallel to the chloroquine technique, which is currently the technique applied at the Hemocentro de Ribeirão Preto. For this, donor red cells samples (n = 50) with known phenotype were sensitized with human antibodies of known specificity and both techniques were applied. The effect of the technique on the expression of erythrocyte antigens (Fya, Fyb, Jka, Jkb, S and s) was also verified by the phenotyping of the samples that had negative DAT. The viability of the antibodies present in the recovered eluate after treatment with EDTA/glycine-acid. A was also tested EDTA/glycine-acid was effective in negatively affecting DAT in 33 samples (66%), comparable to chloroquine that negatived 28 samples (56%). The eluate was viable in treatment with EDTA/glycine-acid and no destruction of erythrocyte antigens after treatment. We conclude that the EDTA/ glycine-acid technique can be used as an option in the treatment of red cells with TDA positive, with the advantage that its execution time is inferior to that of chloroquine (2 minutes x 30 to 120 minutes), which makes it useful in emergency situations. When both techniques are used in different erythrocytes of the same sample, the effectiveness is significantly increased (p <0.05).

A mudança anual de penas é um componente crítico do ciclo biológico de todas as espécies de aves e pode modificar as respostas imunológicas individuais, sendo que suas consequências são pouco estudadas. Neste contexto, uma vez que são escassos os estudos acerca do perfil clínico e parâmetros imunológicos de pinguins-de-Magalhães em diferentes fases da muda, este estudo propôs avaliar determinados aspectos relacionados a esses perfis, em animais mantidos em ambiente controlado. Foram estudados 21 indivíduos adultos (dez machos e 11 fêmeas) e os parâmetros avaliados foram: teste de hipersensibilidade cutânea tardia à fitohemaglutinina (FHA) com a avaliação histopatológica de biópsias que incluíram a análise qualitativa e quantitativa dos tipos celulares presentes na reação e no controle às 6, 24 e 48 horas; parâmetros clínicos (massa corpórea e hematologia); fagocitose e burst oxidativo; fenotipagem e resposta linfoproliferativa. O teste de FHA demonstrou diferença significativa na espessura da membrana interdigital de ambas as patas inoculadas com FHA e com o controle (PBS) quando comparadas antes da inoculação e antes da biópsia às 24 e 48 horas. A análise histomorfométrica revelou não haver diferenças significativas na frequência relativa de granulócitos (heterófilos/ eosinófilos), linfócitos, macrófagos ou trombócitos entre as patas inoculadas com FHA e PBS em nenhum dos momentos analisados. Este resultado sugere que a diferença na espessura da membrana interdigital das patas inoculadas seja em decorrência do edema e não do infiltrado inflamatório. Para analisar os efeitos do ciclo da muda nos parâmetros clínicos e hematológicos, os animais foram divididos em três grupos: pré-muda (-5 a 0 dias da muda), muda (10&ordm; ao 21&ordm; dia da muda) e padrão basal (&gt;120 dias da muda). Os resultados mostraram que em relação ao momento basal: houve aumento significativo na massa corpórea dos animais no momento da pré-muda; diminuição significativa de proteínas totais, hematócrito, eritrócitos totais e leucócitos totais na pré-muda e muda. Não houve diferença no número total de monócitos e heterófilos, contudo, houve eosinofilia e linfopenia na pré-muda e muda, respectivamente. Com relação aos parâmetros imunológicos, houve aumento no burst oxidativo gerado pelo estímulo biológico (Staphylococcus aureus) na pré-muda em relação à muda e ao padrão basal; ocorreu aumento do burst oxidativo gerado pelo estímulo químico (éster de forbol) na pré-muda em relação à muda. Em relação à fagocitose, houve diminuição desta capacidade dos leucócitos dos animais na pré-muda e muda em relação ao padrão basal quando o agente foi bacteriano; entretanto, o mesmo não foi observado para a fagocitose de leveduras (Zymosan A); houve diminuição de linfócitos T CD4+ circulantes na pré-muda e muda em relação ao padrão basal e o índice de proliferação de linfócitos aumentou durante o período de muda em relação ao basal. Esses resultados tomados em conjunto sugerem que as mudanças fisiológicas observadas durante o ciclo da muda interferem na modulação de parâmetros imunológicos e são sugestivas de serem consequência do prejuízo energético sofrido, mesmo em animais mantidos em ambientes controlados / The annual replacement of feathers is a critical component of the biological cycle of all bird species. Molt can modify their individual immune response, and the consequences of this are not well known. Within this context, and the few studies on the clinical profile and immunological parameters of Magellanic penguins at different stages of their molt, this study aimed to evaluate certain aspects related to the clinical profiles of animals kept in a controlled environment. A total of 21 adult individuals (ten males and 11 females) was analyzed and the parameters evaluated in this study were: delayed-type hypersensitivity test to phytohemagglutinin (PHA) based on the histopathological evaluation of foot web biopsies that included qualitative and quantitative analysis of cell types present in the essay and control at 6, 24 and 48 hours; clinical parameters (body mass and hematology); phagocytosis and oxidative burst; phenotyping and lymphoproliferative response. The PHA test showed significant differences in the thickness of the webbing of both feet inoculated with PHA and control (PBS), when compared before inoculation and before biopsy at 24 and 48 hours. Histomorphometric analysis revealed no significant differences in the relative frequencies of granulocytes (heterophils/ eosinophils), lymphocytes, macrophages, thrombocytes, or between the feet inoculated with PHA and with PBS in any of the samples. This result suggests that the difference in thickness of the foot webbing is due to edema and not an inflammatory infiltrate. To analyze the effects of the molting cycle on clinical and hematological parameters, the birds were divided into three groups: pre-molt (from -5 days to the start of the molt, day 0), molt (10th to 21st days of molt) and baseline (&gt;120 days after the completion of the molt). The results showed that in comparison with baseline values: a significant increase in body mass occurred during pre-molt; and a significant decrease in total protein, hematocrit, total erythrocytes and total leukocytes occurred during pre-molt and molt. There was no difference in the total numbers of monocytes and heterophils, however, there were eosinophilia and lymphopenia during pre-molt and molt, respectively. There was an increase in the oxidative burst generated by the biological stimulus (Staphylococcus aureus) in the pre-molt compared to molt and baseline levels; and there was an increased oxidative burst generated by the chemical stimuli (phorbol esters) in the pre-molt in relation to the molt. There was a decreased capacity of phagocytosis for the leukocytes in animals during pre-molt and molt compared to baseline when the agent was bacterial; however, this was not observed for the phagocytosis of yeast (Zymosan A). There was a decrease of circulating CD4+ T lymphocytes in the pre-molt and molt compared to baseline, and the lymphocyte proliferation index increased during the molt compared to baseline. The results suggest that the physiological changes observed during the molt cycle does interfere with the modulation of immune parameters, and are suggestive of resulting as a consequence of from the high energy demands of molt, even for birds kept in controlled environments