Thesis (Ph. D.)--University of Wisconsin--Madison, 1962. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 64-68).

Universiẗat, Diss., 2006--Giessen.

Avian coccidiosis is caused by the intestinal protozoa Eimeria. The parasite's site of infection in the intestine is site specific. Eimeria acervulina mainly affects the duodenum, E. maxima the jejunum, and E. tenella the ceca. Lesions in the intestinal mucosa cause reduced feed efficiency and body weight gain in Eimeria-challenged chickens. My previous studies showed that the growth reduction may be due to changes in expression of digestive enzymes and nutrient transporters in the intestine. This can also lead to diminished intracellular pools of nutrients and inhibit pathogen replication. In this dissertation, further analysis of cellular events was performed. Expression of host defense peptides (HDPs), apoptosis and autophagy related genes were examined in Eimeria challenged broilers. The results showed that upon Eimeria infection, LEAP2 was consistently downregulated in the target tissues, while the avian beta-defensins (AvBDs) showed many variations in expression patterns. Downregulation of LEAP2 may be a mechanism for Eimeria to combat the host defense system, and to promote its survival inside the host cell. The in situ hybridization results showed that LEAP2 was expressed only along the villus in the small intestine and not in the crypt. This is the first time LEAP2 has been localized to epithelial cells of the chicken intestine. Eimeria infection can also induce an anti-apoptotic and anti-autophagy state in the host cells. This condition can be both favorable and unfavorable to parasite survival and replication inside the host cell. A comparison of gene expression between Ross and Eimeria resistant Fayoumi (line M5.1 and M15.2) chickens challenged with Eimeria maxima was conducted. The comparison among different lines of chickens showed differential gene expression patterns in lines with different resistance to Eimeria. The similar body weight reduction indicated that there may not be a significant Eimeria resistant line among the Ross, Fayoumi M5.1 and M15.2 birds. The interaction between Eimeria and the host cell is very complex. Studying the mechanisms behind the changes of gene expression during Eimeria infection may give rise to potential therapeutic targets of coccidiosis. / Ph. D.

Coccidiosis is endemic in the commercial broiler industry and inflicts devastating economic losses to poultry operations. Probiotics may provide a potential alternative to the prophylactic use of anticoccidials in commercial production. This study evaluated the effects of probiotic applications (feed and water) on bird performance and resistance to a mixed Eimeria infection in commercial broilers. On day of hatch, 1008 commercial male broilers (Cobb 500) were assigned to one of 6 treatments (8 replicate floor pens; 21 birds/pen), including non-infected negative control (NEG), Eimeria-infected positive control (POS), anticoccidial control (0.01% salinomycin, SAL), intermittent high dose water-applied probiotic (WPI), continuous low dose water-applied probiotic (WPC), and feed-supplemented probiotic (FSP). On d15, all birds except those in NEG were challenged with a mixed inoculum of Eimeria acervulina, E. maxima, and E. tenella. Measurements were taken on d7, 15, 21, 28, 35 and 42. Fecal samples were collected from d20-d24 for oocyst counts, and lesion scores were evaluated on d21. Data were analyzed using the Fit Model platform in JMP Pro 10.0 (SAS). Differences in experimental treatments were tested using Tukey HSD following ANOVA with significance reported at P ° 0.05. Overall, NEG birds outperformed all other groups. For performance, the probiotic groups were comparable to the SAL treated birds, except during the 6 days immediately following the Eimeria species challenge, where the SAL birds exhibited better performance. WPC birds had lower duodenal and jejunal lesion scores, indicating a healthier intestine and enhanced resistance to Eimeria species compared to POS. Birds in the WPI treatment shed fewer oocysts in the feces, though this was not a trend for all of the probiotic treatment groups. The results of this study suggest probiotic supplementation without anticoccidials can enhance performance and help alleviate the negative effects of a mixed Eimeria infection. / Master of Science

Coccidiosis is a disease caused by protozoa of the genus Eimeria ssp. These protozoa are intracellular parasites of enterocytes that rupture the host cell, causing damage to the intestinal mucosa. The lesions caused by Eimeria reduce nutrient uptake by broilers, affecting their productivity gain, and also represent a portal of entry for other enteropathogens. The aim of this study was to assess the correlation between lesions caused by Eimeria and the prevalence of coccidiosis and other gastrointestinal disorders among broilers reared in Brazil from 2012 to 2014. Intestinal health was evaluated at 82 poultry houses in Brazil, totaling 5,528 birds aged 12 to 40 days. The rearing period was divided into two phases: phase 1 (12 to 21 days) and phase 2 (22 to 40 days). The broilers, at least three per shed, were collected from three different sites. The following gastrointestinal aspects were analyzed in the present study: presence of cell desquamation, excess fluid, excess mucus, ingestion of contaminated litter, thickened intestinal walls, thin intestinal walls, movement of food bolus, abnormal intestinal tonus, Turkish towel appearance, verminosis, and necrotic enteritis. The classification of the scores for gross lesions caused by Eimeria acervulina, Eimeria maxima, and Eimeria tenella followed the method proposed by Johnson & Reid, [8] and the oocyst count of E. maxima (E. maxima micro) in the mucosa was performed under a light microscope at 100X magnification. The statistical analysis of the Pearson correlation coefficient was carried out by the SAS 9.3 software program [16], using a 95% confidence interval. The results of this study revealed that E. acervulina was the most prevalent (mean of 13.5%) species in both rearing stages. Also, there was a positive correlation with thin intestinal walls and abnormal intestinal tonus in phases 1 and 2, as well as a positive correlation with ingestion of contaminated litter in phase 2. The second highest prevalence was that of E. maxima (mean of 6.75%), with a positive correlation with excess mucus, thickened and thin intestinal walls in phase 1, and a positive correlation with cell desquamation, excess fluid, and Turkish towel appearance in phase 2. E. tenella yielded the lowest prevalence rates (mean of 4.35) among the analyzed Eimeria species, showing a positive correlation with excess fluid in phases 1 and 2 and with thickened intestinal walls and lesions caused by E. maxima in phase 2. The microscopic analysis demonstrated that E. maxima was found in 18% of mucosal scrapings in phase 1, which accounts for a subclinical coccidiosis rate of 282.98% compared with clinical coccidiosis. A positive correlation was observed for E. maxima micro between thickened intestinal walls and lesions caused by E. maxima. E. maxima was detected in mucosal scrapings of 29.6% of the broilers in phase 2, accounting for a subclinical coccidiosis rate of 236.37% compared with clinical coccidiosis. E. maxima micro revealed a positive correlation with excess fluid, necrotic enteritis, E. acervulina, E. maxima, and E. tenella in phase 2. The comparison between the rearing periods showed that subclinical coccidiosis affected 64.45% more broilers in phase 2 than in phase 1. In the gross analysis, E. acervulina was the most prevalent species in both rearing periods. A lesion score equal to 1 was the most frequent among all Eimeria species. Subclinical coccidiosis affected a significant number of broilers in the analyzed Brazilian flocks, and was correlated with several factors that reduce intestinal health. It may be concluded that monitoring is of utmost importance to find out the status of intestinal health of poultry. The microscopic detection of E. maxima (mean of 23.8%) is correlated with factors that negatively affect intestinal health. / A coccidiose é uma enfermidade causada por protozoários do gênero Eimeria ssp. Esses são parasitas intracelulares de enterócitos que rompem a célula hospedeira causando lesões na mucosa intestinal. As lesões causadas pelas Eimerias resultam em redução na capacidade de absorção de nutrientes, afetando o ganho produtivo dos frangos de corte, e representam uma porta de entrada para outros enteropatógenos. Sendo assim, o objetivo desse estudo foi analisar a correlação entre as lesões causadas pelas Eimerias, e a prevalência de coccidiose e de demais alterações encontradas no trato gastrointestinal de frangos de corte produzidos, no Brasil, no período de 2012 a 2014. As avaliações da saúde intestinal foram realizadas em 82 integrações de frangos de corte, no Brasil, totalizando 5.528 aves analisadas com idades entre 12 e 40 dias. O período de produção analisado foi dividido em duas fases: 1ª fase (12 aos 21 dias) e 2ª fase (22 aos 40 dias). Os frangos necropsiados foram coletados em três diferentes pontos e no mínimo três aves por galpão. No presente estudo foram analisadas as seguintes alterações do trato gastrintestinal: presença de descamação celular, excesso de fluido, excesso de muco, ingestão de cama, intestino espesso, intestino fino, passagem de alimento, tônus alterado, toalha turca, verminose e enterite necrótica. A definição dos escores macroscópicos de lesão causados pelas Eimeria acervulina, Eimeria maxima, Eimeria tenella seguiram a metodologia de Johnson & Reid [8], e a contagem de oocistos na mucosa para E. maxima (E. maxima micro) foi realizada com auxílio de microscópio óptico com aumento de 100 X. A análise estatística do coeficiente de correlação de Pearson foi feita com o programa SAS 9.3., com intervalo de confiança de 95%. Os resultados desse estudo demonstraram que a espécie E. acervulina foi a que apresentou maior prevalência (média de 13,5%) em ambas as fases de produção avaliadas. Ainda, referente à E. acervulina, observou-se correlação positiva com intestino fino e tônus intestinal alterado na 1ª e 2ª fase, bem como correlação positiva com ingestão de cama apenas na 2ª fase. A segunda maior prevalência foi da espécie E. maxima (média de 6,75%), obteu-se correlação positiva com excesso de muco, intestino espesso e fino na 1a fase e correlação positiva com descamação celular, excesso de fluído e toalha turca na 2a fase avaliada. A E. tenella representou a menor prevalência (média de 4,35) entre as espécies de Eimerias analisadas, apresentando uma correlação positiva na 1ª e 2ª fase com o excesso de fluído e na 2ª fase com o intestino espesso e com lesões de E. maxima. Na avaliação microscópica, a E. maxima esteve presente em 18% dos raspados de mucosa realizados na 1ª fase, o que representa uma coccidiose subclínica de 282,98% com relação a coccidiose clínica. Para a E. maxima micro foi detectada correlação positiva entre os achados com o intestino espesso e com as lesões de E. maxima. Na 2ª fase, E. maxima foi encontrada nos raspados de mucosa de 29,6% das aves, representando uma coccidiose subclínica de 236,37% com relação à coccidiose clínica. A E. maxima micro apresentou na 2ª fase uma correlação positiva com o excesso de fluido, enterite necrótica, E. acervulina, E. maxima e E. tenella. Na análise comparativa entre os períodos, a coccidiose subclínica acometeu 64,45% mais frangos de corte na 2ª fase em relação a 1ª fase. Na avaliação macroscópia de lesões relacionadas à coccidiose, a E. acervulina foi a espécie de maior prevalência em ambas fases de produção. O escore de lesão mais frequente para todas as espécies de Eimerias foi o de grau 1. A coccidiose subclínica acometeu um número expressivo de frangos de corte do plantel brasileiro e foi correlacionada com diversos fatores de diminuição de saúde intestinal. Concluiu-se que o monitoramento é de suma importância para conhecer o status de saúde intestinal dos lotes avícolas. Pois, a E. maxima microscópica está presente (média de 23,8%) com correlação aos fatores que reduzem a saúde intestinal.

The first part of the thesis concerns phylogenetic relationships of eimerian coccidia from arvicoline rodents based on nuclear 18S rRNA, and mitochondrial COI and COIII genes, as well as morphological and morphometrical analyses of the eimerian oocysts. Population-genetic structure of eimerian coccidia was also analyzed and haplotype networks were constructed. The second part of the thesis is based on elucidation of the origin of infections of arvicoline rodents with Isospora spp. using three different approaches - phylogenetic analyses (18S rRNA, COI, and COIII genes), morphological and morphometrical data, and experimental infections. Techniques of field parasitology, laboratory methods of parasitological examination, molecular-genetic diagnostics, phylogeny, and taxonomy were used in the course of both parts of the study.

Orientador: Nilva Kazue Sakomura / Resumo: Doenças entéricas se apresentam como um dos maiores problemas da produção avícola. Historicamente, antibióticos promotores de crescimento (APC’s) têm sido usados para controlar essa situação. No entanto, as preocupações a respeito de resistência bacteriana a antibióticos têm levado a proibições regionais para reduzir o uso de APC’s. Alternativamente, microrganismos administrados diretamente na dieta como por exemplo os probióticos, podem impactar positivamente sobre a microbiota intestinal, prevenindo problemas entéricos que, inevitavelmente culminam em perdas no desempenho das aves. Outro ponto importante a ser considerado quando se fala em desafio sanitário é o consumo de ração dos animais. A utilização de modelos matemáticos que consideram o fator desafio para modelagem do consumo ainda é escasso. Nesse sentido, essa dissertação foi conduzida com dois principais propósitos: 1) investigar os efeitos de Bacillus amyloliquefaciens CECT 5940 como probiótico (DFM) sozinho ou em associação com bacitracina metileno disalicilato (BMD) em frangos de corte sobre desafio de patógeno entérico. 2) modelar o consumo relativo de animais sobre condição de desafio sanitário e integrar esse modelo dentro de um modelo mecanicista de simulação, o Broiler Growth Model (BGM). Assim, permitindo a modelagem do desempenho de animais sob condição de desafio sanitário. Para isso, um total de 1.530 frangos de corte machos Cobb500 com um dia de idade foram casualizados em cinco tratamentos, com nove r... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Enteric diseases appear one of the biggest problems of poultry production. Historically, antibiotics growth promoter (AGPs) have been used to control this framework. However, concerns about antibiotic resistance bacteria have led regional bans for reducing the use of AGPs. Alternatively, microorganisms directly supplemented in diets such as probiotics, may exhibit a positive impact on intestinal microbiota avoiding enteric problems that inevitably causes loss on broilers performance. Another important point to be considered when talking about sanitary challenge is the feed intake. The use of mathematical models that consider the challenge factor for modelling in this feed intake is still scarce. In this sense, this project was conducted with two main purposes: 1) to investigate the effects of Bacillus amyloliquefaciens CECT 5940 as a probiotic (DFM) alone or in combination with bacitracin methylene disalicylate (BMD) in broilers on enteric pathogen challenge. 2) Model the relative feed intake of animals under sanitary challenge condition and to integrate this model into the mechanistic simulation model, the Broiler Growth Model (BGM). Thus, allowing the modelling of performance of animals under sanitary challenge condition. For such, a total of 1,530-day-old male Cobb500 chicks, were randomly assigned to five treatments, with nine replicate pens with 34 birds each. Treatments included positive control (PC, basal diet without additives or challenge); negative control (NC, basa... (Complete abstract click electronic access below) / Mestre

Parasites represent one of the most common ecological strategies and host-parasite coevolution belongs among the major processes governing evolution of biodiversity on the global scale. Genetic structure and diversity of populations of parasites and their hosts, and their genetic connectivity are the key elements in long-term population survival and evolution. Host switches often disturb the parallel evolution of interacting taxa, even in highly host-specific parasites. Evaluation of importance of the degree of intimacy between parasites and hosts is not a trivial task, because evolutionary patterns observed today were formed by an interplay of many (sometimes previously unforeseen) historical and ecological factors. To reveal the mechanisms of coevolution between parasites and their hosts, inter- and intra-specific genealogical structures in three model systems were analyzed: namely, the sucking lice Polyplax serrata and Apodemus hosts, chewing lice of the genus Menacanthus and endoparasitic coccidean genus Eimeria from Apodemus mice.

A total of 176 kangaroo rats (151 Dipodontys ordii and 25 Q• microps) were captured in northwestern utah and examined for coccidia. Of these 176 rats, four Q• ordii (2.6%) and four Q• microps (16.0%) were infected with ~ utahensis, a new species of coccidia. Little seasonal difference was found in the infection rate of either species. The characteristics of the sporulated oocysts of this species were described. A sporocyst plug was reported for the first time in an eimerian oocyst. Artificially excysted sporozoites were studied by various methods. Thirty living sporozoites averaged 22.5 p in length by 4.5 f in width at the anterior refractile body and 4.6 p in width at the posterior refractile body. The refractile body was ellipsoidal and occupied almost half of the sporozoite. The refractile bodies were protein in nature. Living sporozoites exhibited gliding, flexing, pivoting, and probing movements. Subpellicular fibrils, anterior median rod-shaped organelles, and transverse striations of unknown significance were seen in living and stained sporozoites. In the vesicular nucleus the DNA was concentrated at the periphery and three to five chromatin clumps were present. Little, or no, glycogen was present. The test for lipids was inconclusive. The mean prepatent period in experimentally infected D• ordii was 9.8 days. The discharge of oocysts continued for prolonged periods, evidently as a result of reinfection, although concerted efforts were made to prevent this. The asexual endogenous stages were located in epithelial cells in the distal half of the villi of the small intestine. Four generations of schizonts were present. Mature first-generation schizonts were found 2 1/2 days after inoculation of the animals and contained 12 to 16 merozoites. Mature second-generation schizonts were found on the fourth post-inoculation day and also contained 12 to 16 merozoites. Mature t hirdgeneration schizonts were present on the fourth, fifth and sixth postinoculation days and contained 4 to 8 merozoites. The third-generation schizonts gave rise to early sexual stages or to fourth-generation schizonts. Mature fourth-generation schizonts were found on the sixth and seventh post-inoculation days and contained 16 to 24 merozoites. Young gametocytes were first observed on the fifth post-inoculation day. Shortly after the gametocytes entered the infected epithelial cells the cells became displaced into the lamina propria and the mature gametocytes were usually found in the latter location. The nuclei of infected host cells became considerably enlarged and modified in shape and position. In many host cells there appeared to be two or more nuclei in the parasitized cell; this was interpreted as an artifact of sectioning. However, in a few instances young gametocytes were observed in cells in which the host cell nuclei were undergoing division, indicating that some infected host cells might have been multinucleate. Microgametocyte nuclei were randomly arranged in the microgametocyte during the early stages of development. As the microgametocytes approached maturity the nuclei became arranged in whorls at the surface of compartments. At maturity the microgametes lost their whorl arrangement and became randomly arranged around a central mass of residual material. The mature microgametocytes averaged 63 .9 by 48.3 p• The plastic granules of the macrogametes were slightly eosinophilic with hematoxylin and eosin stain, but did not stain with iron hematoxylin. The macrogametes measured 32 .5 by 27 .0 p at the stage in which the plastic granules were at the periphery of the parasite but had not yet coalesced. Eimeria utahensis caused no outward signs of coccidiosis in experimentally infected D• ordii, nor were any marked pathological changes observed in the tissue sections .

The objective of this study was to produce a monoclonal antibody specific for the microgametocytes of Eimeria tenella, examine the site and stage specificity of the antibody, and investigate the immunopotency of the antibody. BALB/c mice were immunized with antigen containing Eimeria tenella microgametocytes isolated from in vitro systems. After three intraperitoneal immunizations with the antigen and one booster immunization administered by tail vein injection, the mice were sacrificed and their spleen cells fused with SP2/0 mouse myeloma cells using polyethylene glycol as a fusing agent. Resultant hybridomas were screened by immunoelectrophoresis, indirect immunofluorescent antibody assay, and immunoelectron microscopy to determine the isotype, subisotype, site and stages pecificity of the antibody. Of four 96 well plates seeded with fusion products, four hybridomas were found to be producing anti body specific for the target antigen. Only the most strongly positive of these hybridomas, clone T1A3B9, was used for the study. The antibody produced by this hybridoma was found to be of sub isotype IgG2b. T1A389 monoclonal antibody was introduced into Eimeria tenella infected cell cultures on days four, five, and six post-infection. At seven days post-infection, oocyst production was assayed by fixing, staining, and counting the resultant oocysts. Results of the in vitro experiments showed a greater than 50X reduction in oocyst product ion in experimental cultures over controls. Statistical significance of the data were confirmed by a Mann-Whitney U Test. These results indicate that the monoclonal a ntibod y was exert ing an inhibitory effect on the fertilization process. T1A3B9 monoclonal antibody was incubated with Eimeria tenella infected cecal scrapings and cell culture material, immunolabeled with colloidal gold conjugates, and observed by electron microscopy. Results showed that the antibody was binding to the microgametocytes and to no other life cycle stages of the parasite, nor was it binding to host tissue. This indicates that the antibody is stage specific. Additionally, the antibody was seen to bind only to areas in close proximity to the budding flagella of developing microgametes, thus indicating distinct site specificity.