Thesis (Ph. D.)--Auburn University, 2008. / Abstract. Includes bibliographical references.

Thesis (Ph.D)--Murdoch University, 2009. / Thesis submitted to the Faculty of Health Sciences. Includes bibliographical references (leaves 150-162)

A comparative study of the parasites Cryptosporidium andersoni and Giardia duodenalis in feedlot cattle in Western Australia (n=502) and Alberta, Canada (n=852) was conducted. The objectives were to determine the prevalence, infection patterns and impact on cattle performance of these protozoan parasites. Utilizing molecular tools G. duodenalis was genotyped and C. andersoni samples were confirmed positive. C. parvum was absent from all cattle sampled in Alberta, Canada and Western Australia, likely due to the advanced age of the cattle being sampled (6-36 months of age). No C. bovis or C. ryanae were observed in the study cattle. C. andersoni was present in 25% of the groups of feedlot cattle sampled in Western Australia with a prevalence range of 0-26% and in all 3 of the Alberta, Canada study groups with a prevalence range of 2.9-12%. All three Alberta, Canada studies collected performance data, however, there was no significant difference between infected and non-infected steers’ ADG in the feedlot. G. duodenalis was present in 83% of the groups sampled in Western Australia with prevalence ranging from 0–22% and all three study groups sampled in Alberta, Canada were positive with a prevalence ranging from 39–82%. The prevalence of G. duodenalis is significantly higher in the Alberta, Canada studies as compared to the Western Australia studies, probably due to climatic factors. Molecular characterization of a small number of the Alberta, Canada G. duodenalis positive samples (10) revealed 30% (3) genotype A, and 70% (7) genotype E. The same characterization of the Western Australia samples (10) showed 20%(2) genotype A, 40% (4) genotype E, 10% (1) genotype B, 10% (1) genotype C, 10% (1) genotype D and 10% (1) genotype B and E. Due to the unusual finding of genotypes C and D in cattle on such a small number of samples this result should be further studied to either confirm or refute the existence of genotypes C and D in cattle. Based on these results 30% of the animals from Alberta, Canada have the potential to be zoonoti (genotypes A and B) and 40% from the Western Australia studies. The results of this study demonstrate that C. andersoni and G. duodenalis are prevalent in the study feedlot cattle in Western Australia and Alberta, Canada however the impact of these parasites was not negative on animal performance in the Alberta, Canada studies where it was measured.

Giardia and Cryptosporidium are protozoan parasites known to cause enteric disease in terrestrial mammals, reptiles and birds. Compared to the abundance of surveys that have examined Giardia and Cryptosporidium in terrestrial wildlife species, very few studies on either parasite have been undertaken on marine mammal species. Studies of shellfish, marine waters and water treatment plants clearly indicate that marine ecosystems are contaminated with Giardia and Cryptosporidium. In spite of these data the extent to which these parasites extend into the marine environment and how they may impact on marine mammal health remains largely unknown. The aim of this thesis was to expand our current knowledge of Giardia and Cryptosporidium in the marine environment and in particular, the harp and hooded seal populations of the Gulf of St. Lawrence, Canada. A large-scale serological survey of a large cohort of serum samples clearly show that, as is the case with terrestrial mammals, Giardia is ubiquitous in the marine environment. Sera positive for G. duodenalis-specific IgG were detected in almost all cetacean and pinniped species examined, and from all regions of the St. Lawrence estuary, Gulf of St. Lawrence and from the Canadian arctic. In the case of harp and hooded seals, they are actively infected with Assemblage A, a zoonotic strain of G. duodenalis and represent a previously unrecognised contributor to the overall environmental parasite burden. The discovery of this variant of Giardia in a phocid host, along with their susceptibility to infection with terrestrial strains of both Giardia and Cryptosporidium, highlight the potential zoonotic transmission from seals to humans through the consumption of uncooked intestines and general animal handling during research or hunting practices. The identification of this zoonotic strain of Giardia in seals also demonstrates the potential for anthropogenic activities such as human sewage treatment and agriculture runoff to be a significant source of contamination for marine mammals.

Thesis (Ph. D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1989. / Includes bibliographical references (leaves 183-228).

Thesis (M.S.)--University of Massachusetts Amherst, 2009. / Includes bibliographical references (p. 81-93).

Orientador: João Aristeu da Rosa / Banca: Herminia Yohko Kanamura / Banca: Semiramis Guimarães Ferraz Viana / Banca: Vera Lucy de Santi Alvarenga / Banca: Lúcia Pagliusi Castilho / Resumo: Devido à baixa sensibilidade do exame parasitológico de fezes (EPF) para o diagnóstico laboratorial da giardíase, o método imunoenzimático (ELISA – Enzyme – linked immunosorbent assay) para pesquisa de coproantígenos de Giardia tem sido utilizado, mostrando alta sensibilidade e especificidade. Este trabalho foi desenvolvido com o objetivo de se avaliar a eficácia do ELISA ProSpecT Giardia (Alexon, Inc. BioBrás) anti-GSA65 (antígeno fecal específico de Giardia - peso molecular 65.000 Da) como instrumento de controle de cura da giardíase e o seu limiar de positividade. A contagem de cistos por grama de fezes foi feita utilizando a câmara de Neubauer em amostras positivas para G.lamblia de 100 pacientes. Os resultados mostraram eliminação de 3,6 x 106 cistos por grama de fezes em média. Em gráfico esses resultados desenharam uma curva assimétrica positiva, sugerindo a ocorrência de falso-negativos. Avaliou-se também a eficácia do ELISA anti-GSA65 e o método de Faust e Cols (MFc) em detectar mínimas concentrações de cistos de G. lamblia em amostras fecais. Fezes positivas foram diluídas em fezes negativas de modo a se obter amostras com 100, 1.000 e 10.000 cistos por grama de fezes. O ELISA anti-GSA65 mostrou uma positividade significativamente maior que o MFc. Amostras contendo 10.000 cistos por grama de fezes apresentaram 84,7% de positividade pelo ELISA anti-GSA65 e 34,7% pelo MFc. A eficácia do ELISA anti-GSA65, como instrumento de controle de cura após o tratamento pelo metronidazol em 91 pacientes com giardíase, foi avaliada e mostrou que o ELISA anti-GSA65 pode ser usado para monitorar a cura de pacientes utilizando apenas uma amostra fecal, coletada entre o 4º e 7º dias após o tratamento. Pelo MFc seriam necessárias a coleta de duas ou três amostras. Foi avaliada também comparativamente a eficácia do ELISA anti-GSA65 e do... (Resumo completo, clicar acesso eletrônico abaixo). / Abstract: Due to the low sensitivity of the fecal examination (EPF) in detecting cysts of Giardia lamblia, the imunoenzimatic assay (ELISA enzyme linked immunosorbent assay) to detect G.lamblia coproantigens have been used and have showed high sensibility and specificity. With the ain of evaluating the efficacy of ELISA ProSpecT Giardia (Alexon, Inc. BioBrás) anti-GSA65 (Giardia stool specific antigens - molecular weigh 65.000 Da) as monitoring of the cure of giardiasis and its threshold of positivity it was developed this research. The cysts count per grama of faeces was calculated using Neubauer camera in positive fecal samples for Giardia in 100 patients. It was found a concentration of 3.6 X 106 cyst per grama of faeces as average. In a graph the data draw an assymmetrical positive curve, suggesting occurrence of false negative results. It was evaluated the efficacy of ELISA anti-GSA65, and fecal examination by Faust and colls. procedure (MFc), in detecting minimum concentrations of Giardia cysts in fecal samples. To obtain samples with 100, 1.000 and 10.000 cysts per grama, positive samples were diluted in negative samples. ELISA showed a positivity significative higher than MFc. Samples with 10.000 cystis per grama showed 84,7% of positivity by ELISA anti GSA65 and 34,7% by MFc. The efficacy of ELISA anti-GSA65 as cure monitoring after treatment with metronidazole in 91 patients with giardiasis, was evalued and showed that ELISA anti-GSA65 can be used to monitor the cure of patients, using only single sample of each patients, collected between the 4th and 7th days after treatment, whereas with MFc it would be necessary the exam of two or tree samples. It was also evaluated the efficacy of ELISA-anti-GSA65 and MFc in fecal samples of 96 patients. Considering 38 Giardia true positives and 58 true negatives, the ELISA anti-GSA65 showed 100% of sensibility... (Complete abstract, click electronic address below). / Doutor

In the present study methods which can be integrated into a complete lab on a chip system for the detection and characterisation of Giardia duodenalis cysts and Cryptosporidium spp. oocysts from foods were developed and tested. Microfluidic chips, which make use of inertial separation, were designed and fabricated for the concentration and separation of either cysts or oocysts from food particles. These chips were highly specific for their intended target and were shown to be effective when used for artificially contaminated lettuce samples. The quantification by real-time PCR of Cryptosporidium spp. hsp70 mRNA, expressed in response to a heat stress, was assessed as a potential lab on a chip method for the detection of viable oocysts from foods. This method proved to be effective in determining the viability of oocysts in apple cider and the effects of high hydrostatic pressures on the viability of oocysts.

I. A number of commercial enzyme immunoassay (EIA) tests are available for the diagnosis of giardiasis. In a time of rising health-care costs, there is a need for diagnostic tests that are rapid, specific, sensitive and inexpensive. In the first phase of this study, I developed a monoclonal-based EIA, the GIARDIA TEST, with these qualities in mind. This assay's performance characteristics were determined by a comparison study using conventional ova and parasite examination, immunofluorescence antibody test (IFA) and other commercial EIA tests. Studies were done in-house at TechLab, Inc. and at various U.S. medical facilities. Results were statistically analyzed to determine sensitivity (ability of the assay to detect a positive result), specificity (amount of cross-reactivity), predictive positive value (the confidence in a positive result), predictive negative value (the confidence in a negative result) and overall correlation with the reference assay. II. There remain many questions to be answered about the various antigens produced by Giardia lamblia and how they can be utilized as diagnostic markers. In the second phase of this study, I identified and partially characterized the antigen (Ct7 Ag) that reacts with the Ct7 monoclonal antibody (MAb). This MAb is an IgM class mouse immunoglobin that is utilized by the GIARDIA TEST and by an immunofluorence antibody test (IFA) which detects Giardia cysts in water and feces. The results of this study will provide physicians and researchers with detailed information about the Ct7 Ag and why it is a useful marker for giardiasis. / Master of Science

Giardia intestinalis is a protozoan parasite that causes diarrhea, and due to its 200 million human cases each year it has become important to try and reduce the infected. Giardia intestinalis expresses so called variant surface proteins (VSPs) on its surface. VSPs are surface antigens that the immune system can detect and react to. In order to hide from the immune system and protect itself Giardia is able to perform antigenic variation, where it switches the expressed VSP to another one of the 200 VSPs in its genomic repertoire. Understanding the regulation and expression of the VSPs is an important steppingstone towards being able to create a vaccine or a better treatment to try to reduce the negative health effects created by this parasite. The main components that regulate VSPs that are being expressed on the surface are Dicer and Argonaute. They are both involved in the RNA interference (RNAi) machinery that has been suggested to silence the VSPs that are not being expressed post-transcriptionally. The aim of this study is to create mutants of Giardia that will no longer express Argonaute or Dicer and thereby be able to analyze the VSP expression and to see if a difference in expression can be detected. This can lead to an understanding of the regulatory mechanism of antigenic variation. We were able to create mutants that most likely did not express Argonaute or Dicer, however since PCR verification of successful knockout did not work, except for mutant Dicer nr. 12, genome sequencing is needed for verification of all the knockout mutants. When analyzing the VSP expression using immunostaining the only surviving Argonaute mutant and the two Dicer mutants that had gone through an initial limiting dilution showed that there was a significant difference in VSP expression for the Argonaute mutants and one of the Dicer mutants. There was a higher fluorescence detected in the nr. 8 Argonaute mutant and the 12#2 Dicer mutant, which indicate a higher VSP expression for those two strains compared to the Cas9 expressing wild type. Furthermore, fluorescence dots could be detected around the parasites especially for Dicer 12#2 which could indicate that this mutant has a higher turnover of VSPs and is releasing more of them into the surrounding area. Due to time limitations many follow up experiments were not able to be performed, however the results obtained give an important insight into the regulation of VSPs in Giardia intestinalis.